Publications by year
In Press
ffrench-Constant R (In Press). Hybrid effects in field populations of the African Monarch Danaus chrysippus. Biological Journal of the Linnean Society
Yvon-Durocher G, Padfield D, Buckling A, Lowe C, Ffrench-Constant R, Schaum E (In Press). Metabolic compensation constrains the temperature dependence of gross primary production. Ecology Letters
Rhodes M, Bennie J, Spalding A, ffrench-Constant R, Maclean I (In Press). Recent advances in the remote sensing of insects. Biological Reviews
2023
Rutagarama VP, Ireri PM, Sibomana C, Omufwoko KS, Martin SH, Ffrench-Constant RH, Eckardt W, Kaplin BK, Smith DAS, Gordon I, et al (2023). African Queens find mates when males are rare.
Ecol Evol,
13(4).
Abstract:
African Queens find mates when males are rare.
In butterflies and moths, male-killing endosymbionts are transmitted from infected females via their eggs, and the male progeny then perish. This means that successful transmission of the parasite relies on the successful mating of the host. Paradoxically, at the population level, parasite transmission also reduces the number of adult males present in the final population for infected females to mate with. Here we investigate if successful female mating when males are rare is indeed a likely rate-limiting step in the transmission of male-killing Spiroplasma in the African Monarch, Danaus chrysippus. In Lepidoptera, successful pairings are hallmarked by the transfer of a sperm-containing spermatophore from the male to the female during copulation. Conveniently, this spermatophore remains detectable within the female upon dissection, and thus, spermatophore counts can be used to assess the frequency of successful mating in the field. We used such spermatophore counts to examine if altered sex ratios in the D. chrysippus do indeed affect female mating success. We examined two different field sites in East Africa where males were often rare. Surprisingly, mated females carried an average of 1.5 spermatophores each, regardless of male frequency, and importantly, only 10-20% remained unmated. This suggests that infected females will still be able to mate in the face of either Spiroplasma-mediated male killing and/or fluctuations in adult sex ratio over the wet-dry season cycle. These observations may begin to explain how the male-killing mollicute can still be successfully transmitted in a population where males are rare.
Abstract.
Author URL.
Traut W, Sahara K, ffrench-Constant RH (2023). Lepidopteran Synteny Units (LSUs) reveal deep conservation of macrosynteny in butterflies and moths.
Traut W, Sahara K, ffrench-Constant RH (2023). Lepidopteran Synteny Units reveal deep chromosomal conservation in butterflies and moths. G3: Genes, Genomes, Genetics, 13(8).
Grant C, Singh KS, Hayward A, Hunt BJ, Troczka BJ, Pym A, Ahn S-J, Zeng B, Gao C-F, Leroux A, et al (2023). Overexpression of the UDP-glycosyltransferase UGT34A23 confers resistance to the diamide insecticide chlorantraniliprole in the tomato leafminer, Tuta absoluta.
Insect Biochem Mol Biol,
159Abstract:
Overexpression of the UDP-glycosyltransferase UGT34A23 confers resistance to the diamide insecticide chlorantraniliprole in the tomato leafminer, Tuta absoluta.
The tomato leafminer, Tuta absoluta, is an invasive crop pest that has evolved resistance to many of the insecticides used for its control. To facilitate the investigation of the underpinning mechanisms of resistance in this species we generated a contiguous genome assembly using long-read sequencing data. We leveraged this genomic resource to investigate the genetic basis of resistance to the diamide insecticide chlorantraniliprole in Spanish strains of T. absoluta that exhibit high levels of resistance to this insecticide. Transcriptomic analyses revealed that, in these strains, resistance is not associated with previously reported target-site mutations in the diamide target-site, the ryanodine receptor, but rather is associated with the marked overexpression (20- to >100-fold) of a gene encoding a UDP-glycosyltransferase (UGT). Functional expression of this UGT, UGT34A23, via ectopic expression in Drosophila melanogaster demonstrated that it confers strong and significant resistance in vivo. The genomic resources generated in this study provide a powerful resource for further research on T. absoluta. Our findings on the mechanisms underpinning resistance to chlorantraniliprole will inform the development of sustainable management strategies for this important pest.
Abstract.
Author URL.
Penkova E (2023). The Evolutionary Ecology of Antibiotic Resistance in an Insect Model.
Abstract:
The Evolutionary Ecology of Antibiotic Resistance in an Insect Model
The fitness consequences of antibiotic resistance are commonly quantified in vitro, and fitness estimated from growth rate measurements or direct competition experiments in drug-free media, overlooking environmental factors within a live infection which may also affect bacterial fitness, such as colonisation rate, transmission potential, or the presence of other interacting microbes.
This dissertation aimed to assess the effects of both in vitro and in vivo conditions on bacterial fitness post-acquiring spontaneous resistance mutation and validate earlier studies in nutrient rich media or other laboratory systems in live infections. To achieve this, I utilised the larvae of the diamondback moth, Plutella xylostella, and its natural gut symbiont, the opportunistic pathogen Enterobacter cloacae. This experimental system provided novel means of exploring multiple components of fitness in the presence and absence of antibiotics including within-host competitive ability and between-host transmissibility.
First, I generated and characterised a collection of E. cloacae mutants, selected on either cefotaxime, nalidixic acid, or rifampicin and explored the pleiotropic fitness costs associated with resistance. Fitness parameters were quantified in vivo and compared to measurements in vitro. Results suggest that bacterial competitiveness can be environment-dependent, with costs generally enhanced in vivo, highlighting that in vitro measurements may be an unreliable basis for antimicrobial resistance management.
Next, I explored the sociality of antibiotic resistance as a potential mechanism explaining the co-occurrence of sensitive and resistant strains under varying demographic and environmental conditions. The relative fitness of the susceptible strain was measured in broth and biofilms in vitro but also in vivo. Cooperative protection in mixed genotype infections allowed the persistence of susceptible bacteria under selection, conferring fitness benefits when susceptible bacteria were rare. Frequency-dependent fitness suggests stable maintenance of both genotypes, despite resistance costs. Overall these results emphasise the value of exploring diverse fitness components of microbes and illustrate how insect models can provide valuable systems for testing and refining hypotheses on the fitness consequences of resistance mutations.
Abstract.
Orteu A, Kucka M, Katili E, Ngumbao C, Gordon IJ, Ng’iru I, van der Heijden E, Talavera G, Warren IA, Collins S, et al (2023). Transposable element insertions are associated with Batesian mimicry in the pantropical butterfly Hypolimnas misippus.
ffrench-Constant RH (2023). Transposable elements and xenobiotic resistance. Frontiers in Insect Science, 3
2022
Mowbray S, Bennie J, Rhodes MW, Smith DAS, ffrench-Constant RH (2022). Back to the Meadow Brown: eyespot variation and field temperature in a classic butterfly polymorphism.
Kim K-W, De-Kayne R, Gordon IJ, Omufwoko KS, Martins DJ, Ffrench-Constant R, Martin SH (2022). Stepwise evolution of a butterfly supergene via duplication and inversion.
Philos Trans R Soc Lond B Biol Sci,
377(1856).
Abstract:
Stepwise evolution of a butterfly supergene via duplication and inversion.
Supergenes maintain adaptive clusters of alleles in the face of genetic mixing. Although usually attributed to inversions, supergenes can be complex, and reconstructing the precise processes that led to recombination suppression and their timing is challenging. We investigated the origin of the BC supergene, which controls variation in warning coloration in the African monarch butterfly, Danaus chrysippus. By generating chromosome-scale assemblies for all three alleles, we identified multiple structural differences. Most strikingly, we find that a region of more than 1 million bp underwent several segmental duplications at least 7.5 Ma. The resulting duplicated fragments appear to have triggered four inversions in surrounding parts of the chromosome, resulting in stepwise growth of the region of suppressed recombination. Phylogenies for the inversions are incongruent with the species tree and suggest that structural polymorphisms have persisted for at least 4.1 Myr. In addition to the role of duplications in triggering inversions, our results suggest a previously undescribed mechanism of recombination suppression through independent losses of divergent duplicated tracts. Overall, our findings add support for a stepwise model of supergene evolution involving a variety of structural changes. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.
Abstract.
Author URL.
Shanks K, Ffrench-Constant R (2022). Understanding the anti-reflective glasswing butterfly for enhanced solar concentrator optics. Light in Nature IX.
2021
Grant C (2021). Combating insecticide resistance in the tomato leafminer, Tuta absoluta.
Abstract:
Combating insecticide resistance in the tomato leafminer, Tuta absoluta.
BACKGROUND: the tomato leafminer, Tuta absoluta is a damaging pest of tomato crops worldwide. In the UK, T. absoluta is controlled using an integrated pest management (IPM) strategy that includes the pesticides spinosad and chlorantraniliprole, the biocontrol agent Macrolophus pygmaeus and pheromone-based mating disruption. Some growers have reported a loss of efficacy of this technology. There are concerns that T. absoluta may have evolved resistance to these applied chemistries as well as undergone adaptations in its capacity to reproduce asexually. In this thesis I investigate whether pesticide resistance is present in UK populations and identify the molecular mechanisms for this resistance. I will also investigate the capacity T. absoluta to reproduce asexually through parthenogenesis in the absence of males.
RESULTS: I demonstrate that UK populations of T. absoluta are highly resistant to spinosad and identify two novel mechanisms by which resistance has evolved. Analysis of messenger RNA encoding the target site of spinosad, the nicotinic acetylcholine receptor (nAChR) α6 subunit, revealed resistant strains lack exon 4 resulting in a highly truncated protein. In a second resistant strain the deletion of three amino acids is detected in the transmembrane domain of the nAChR - predicted to be the binding site of spinosad. I identify low levels of tolerance to chlorantraniliprole in UK populations and show this resistance can be selected for to produce highly resistant populations. Analysis of the target site of chlorantraniliprole, the ryanodine receptor, identified amino acid substitution G4903V that has been strongly linked to diamide resistance in a range of lepidopteran species including T. absoluta. With regards asexual reproduction, I observed a small but significant increase in the rate of asexual reproduction. This allows persistence of the pest in the presence of the mating disruptor, Isonet T. Marked differences in several other life history traits associated with reproduction were also observed in these populations including increased longevity further allowing T. absoluta’s persistence within the crop.
CONCLUSION: My findings show that the evolution of resistance has rendered spinosad redundant at most sites in the UK. The mechanisms identified are unique to UK populations and so have likely evolved under selection in the UK. Chlorantraniliprole remains effective, however our findings of resistance at low frequency suggest that continued use of this pesticide must be monitored carefully. The low overall occurrence of asexual reproduction observed in this study is unlikely to result in loss of efficacy of mating disruption as reproductive rate remained low. However, the observed changes in longevity and egg laying may allow T. absoluta to persist for longer within the crop, and, together with the increased frequency of parthenogenesis, may reflect selection from the use of Isonet T. Thus, regular monitoring of the reproductive capacity of UK populations should be conducted, along with continual assessment of resistance allele frequencies of pesticides to inform resistance management strategies.
Abstract.
McLeman A (2021). Developing screening tools to identify novel, resistance breaking pesticides.
Abstract:
Developing screening tools to identify novel, resistance breaking pesticides.
Pesticide resistance is estimated to cost the USA $1.4 billion annually. Not only is there a huge economic cost, but the loss of crop yield and higher doses of pesticides needed to control pests damages the ecosystem 1,2. The development of resistance to chemicals is a universal phenomenon and within insect pests more than 440 species are now resistant to one or more pesticidal compound 3,4. As increasing levels of resistance arise and new molecular tools become available the understanding of resistance mechanisms grows and the limitations of pesticides are clarified 5,6. Understanding resistance is vital to counter it 5,7,8.
Still facing high levels of pesticide resistance and the damaging effects of the remaining effective compounds, I here look to identify a novel pesticidal compound to overcome current resistance mechanisms 9. Synthetic compounds made by industrial partner Darr House M.I. were tested for activity against Drosophila melanogaster and Myzus persicae. The first 18 compounds were expected to act on the nicotinic acetylcholine receptor using imidacloprid as a positive control. Four competitively active compounds were found but, following a ban on neonicotinoids in the EU in 2018 and a knock-on lack of interest on the part of major agrochemical companies in novel nAChR compounds, this part of the project was pursued no further 10,11. The next 30 compounds were then tested for activity against the neurotransmitter gamma-aminobutyric acid (GABA) receptor. Here activity was only found against Drosophila not Myzus. Five compounds showed activity against D. melanogaster susceptible strain Canton-S, four then showed activity against metabolic resistant strain Hikone-R with compound 47 being close to resistance breaking.
While synthetic compounds are popular, natural sources are not only a source of inspiration for synthetic products but natural products used for pest control have advantages of being environmentally friendly and constantly evolving with their pests. I tested 9 botanical sources for insecticidal and repellent activity against D. melanogaster and the Peach potato aphid M. persicae. Extracts from samples were taken using a methanol extraction technique. Rosemary extract results suggest potential lethal effects on Drosophila but development of this product would be required to concentrate the lethal effects above 40%. All extracts: basil, chilli, garlic, lemongrass, nasturtium leaves, flowers and seeds and rosemary showed repellent activity against Myzus except dill extract which had no effect. An increase in nymph droppings was seen for Myzus treated with basil suggesting possible problems for use of this compound as aphid control.
To address the problem of cost and identification of novel active pesticides a Fly-Tox panel was developed using D. melanogaster as a model screening tool containing metabolic P450 resistance genes from multiple economically important pests and pollinator species. In this thesis four lines were developed containing Cyp6cm1, Cyp6bq23, Cyp6bq9 and Cyp337b3 but conferral of resistance was unsuccessful. Alternative lines from the published Fly-Tox panel were used to test the use of the screening tool with novel insecticides from chapter 2 and 3; one nAChR and one GABA targeting compound. These novel compounds were compared against positive controls; imidacloprid and fipronil, and showed a successful test run of a section of the screening tool.
No resistance breaking bee-safe compounds were identified in this thesis but there was a successful trial of the Fly-Tox screening tool of transgenic Drosophila showing the value of this new resource in pesticidal discovery science.
There were also findings of broad metabolic capabilities of the gene Cyp6er1, known to metabolise neonicotinoids, but also found to be active against suspected GABA targeting novel compound 47.
Abstract.
Singh KS, De-Kayne R, Omufwoko KS, Martins DJ, Bass C, ffrench-Constant R, Martin SH (2021). Genome assembly of Danaus chrysippus and comparison with the Monarch Danaus plexippus.
Singh KS, De-Kayne R, Omufwoko KS, Martins DJ, Bass C, ffrench-Constant R, Martin SH (2021). Genome assembly of Danaus chrysippus and comparison with the Monarch Danaus plexippus. G3: Genes, Genomes, Genetics, 12(3).
Singh KS, Cordeiro EMG, Troczka BJ, Pym A, Mackisack J, Mathers TC, Duarte A, Legeai F, Robin S, Bielza P, et al (2021). Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.
Commun Biol,
4(1).
Abstract:
Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.
The aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host-plant associations, uncovering the widespread co-option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.
Abstract.
Author URL.
Kim K-W, De-Kayne R, Gordon IJ, Omufwoko KS, Martins DJ, ffrench-Constant R, Martin SH (2021). Stepwise evolution of a butterfly supergene via duplication and inversion.
2020
Singh KS, Hosken DJ, Wedell N, Ffrench-Constant R, Bass C, Baxter S, Paszkiewicz K, Sharma MD (2020). De Novo Genome Assembly of the Meadow Brown Butterfly, Maniola jurtina.
G3 (Bethesda),
10(5), 1477-1484.
Abstract:
De Novo Genome Assembly of the Meadow Brown Butterfly, Maniola jurtina.
Meadow brown butterflies (Maniola jurtina) on the Isles of Scilly represent an ideal model in which to dissect the links between genotype, phenotype and long-term patterns of selection in the wild - a largely unfulfilled but fundamental aim of modern biology. To meet this aim, a clear description of genotype is required. Here we present the draft genome sequence of M. jurtina to serve as a founding genetic resource for this species. Seven libraries were constructed using pooled DNA from five wild caught spotted females and sequenced using Illumina, PacBio RSII and MinION technology. A novel hybrid assembly approach was employed to generate a final assembly with an N50 of 214 kb (longest scaffold 2.9 Mb). The sequence assembly described here predicts a gene count of 36,294 and includes variants and gene duplicates from five genotypes. Core BUSCO (Benchmarking Universal Single-Copy Orthologs) gene sets of Arthropoda and Insecta recovered 90.5% and 88.7% complete and single-copy genes respectively. Comparisons with 17 other Lepidopteran species placed 86.5% of the assembled genes in orthogroups. Our results provide the first high-quality draft genome and annotation of the butterfly M. jurtina.
Abstract.
Author URL.
McLeman A, Troczka BJ, Homem RA, Duarte A, Zimmer C, Garrood WT, Pym A, Beadle K, Reid RJ, Douris V, et al (2020). Fly-Tox: a panel of transgenic flies expressing pest and pollinator cytochrome P450s.
Pesticide Biochemistry and Physiology,
169Abstract:
Fly-Tox: a panel of transgenic flies expressing pest and pollinator cytochrome P450s
There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions.
Abstract.
ffrench-Constant R, Martin S, Bass C, Singh K, Traut W, Gordon I, Smith D, Martins D (2020). Whole-chromosome hitchhiking driven by a male-killing endosymbiont. PLoS Biology
2019
Smith DAS, Traut W, Martin SH, Ireri P, Omufwoko KS, ffrench-Constant R, Gordon IJ (2019). Neo Sex Chromosomes, Colour Polymorphism and Male-Killing in the African Queen Butterfly, Danaus chrysippus (L.).
Insects,
10(9), 291-291.
Abstract:
Neo Sex Chromosomes, Colour Polymorphism and Male-Killing in the African Queen Butterfly, Danaus chrysippus (L.)
Danaus chrysippus (L.), one of the world’s commonest butterflies, has an extensive range throughout the Old-World tropics. In Africa it is divided into four geographical subspecies which overlap and hybridise freely in the East African Rift: Here alone a male-killing (MK) endosymbiont, Spiroplasma ixodetis, has invaded, causing female-biased populations to predominate. In ssp. chrysippus, inside the Rift only, an autosome carrying a colour locus has fused with the W chromosome to create a neo-W chromosome. A total of 40–100% of Rift females are neo-W and carry Spiroplasma, thus transmitting a linked, matrilineal neo-W, MK complex. As neo-W females have no sons, half the mother’s genes are lost in each generation. Paradoxically, although neo-W females have no close male relatives and are thereby forced to outbreed, MK restricts gene flow between subspecies and may thus promote speciation. The neo-W chromosome originated in the Nairobi region around 2.2 k years ago and subsequently spread throughout the Rift contact zone in some 26 k generations, possibly assisted by not having any competing brothers. Our work on the neo-W chromosome, the spread of Spiroplasma and possible speciation is ongoing.
Abstract.
Spalding A, Shanks K, Bennie J, Potter U, Ffrench-Constant R (2019). Optical Modelling and Phylogenetic Analysis Provide Clues to the Likely Function of Corneal Nipple Arrays in Butterflies and Moths.
Insects,
10(9).
Abstract:
Optical Modelling and Phylogenetic Analysis Provide Clues to the Likely Function of Corneal Nipple Arrays in Butterflies and Moths.
The lenses in compound eyes of butterflies and moths contain an array of nipple-shaped protuberances, or corneal nipples. Previous work has suggested that these nipples increase light transmittance and reduce the eye glare of moths that are inactive during the day. This work builds on but goes further than earlier analyses suggesting a functional role for these structures including, for the first time, an explanation of why moths are attracted to UV light. Using a phylogenetic approach and 3D optical modelling, we show empirically that these arrays have been independently lost from different groups of moths and butterflies and vary within families. We find differences in the shape of nipples between nocturnal and diurnal species, and that anti-glow reflectance levels are different at different wave-lengths, a result thereby contradicting the currently accepted theory of eye glow for predator avoidance. We find that there is reduced reflectance, and hence greater photon absorption, at UV light, which is probably a reason why moths are attracted to UV. We note that the effective refractive index at the end of the nipples is very close to the refractive index of water, allowing almost all the species with nipples to see without distortion when the eye is partially or completely wet and providing the potential to keep eyes dry. These observations provide a functional explanation for these arrays. of special interest is the finding that their repeated and independent loss across lepidopteran phylogeny is inconsistent with the explanation that they are being lost in the 'higher', more active butterflies.
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Author URL.
Grant C, Jacobson R, Ilias A, Berger M, Vasakis E, Bielza P, Zimmer CT, Williamson MS, Ffrench-Constant RH, Vontas J, et al (2019). The evolution of multiple-insecticide resistance in UK populations of tomato leafminer, Tuta absoluta.
Pest Manag Sci,
75(8), 2079-2085.
Abstract:
The evolution of multiple-insecticide resistance in UK populations of tomato leafminer, Tuta absoluta.
BACKGROUND: the tomato leafminer, Tuta absoluta, is an economically important pest of tomatoes in Europe, Africa, Asia and South America. In the UK this species is controlled using an integrated pest management (IPM) programme which incorporates the insecticides spinosad and chlorantraniliprole. In response to UK grower concerns of loss of efficacy of these compounds at certain sites, insecticide bioassays were performed on five populations collected from four commercial glasshouses and potential mechanisms of resistance investigated. RESULTS: We observed high levels of resistance to spinosad in four of the strains, and in two of these tolerance to chlorantraniliprole. Selection of one of these strains with chlorantraniliprole rapidly resulted in a line exhibiting potent resistance to this compound. Sequencing of messenger RNA encoding the nicotinic acetylcholine receptor (nAChR) α6 subunit, target of spinosad, revealed Taα6 transcripts in the spinosad-resistant strains that lack exon 4 and encode a highly truncated protein, or contain a triplet deletion in the predicted first transmembrane domain resulting in the loss of a highly conserved amino acid. Sequencing of the ryanodine receptor gene, encoding the target of diamide insecticides, of the chlorantraniliprole-selected line revealed an amino acid substitution (G4903V) that has been previously linked to diamide resistance in populations of T. absoluta in the Mediterranean and South America. CONCLUSION: Taken together our results reveal emerging resistance in UK populations of T. absoluta to two of the most important insecticides used as part of IPM, with significant implications for the control of this species in the UK. © 2019 Society of Chemical Industry.
Abstract.
Author URL.
Manktelow J (2019). Virulence and Evolutionary Ecology in the Entomopathogen Bacillus thuringiensis.
Abstract:
Virulence and Evolutionary Ecology in the Entomopathogen Bacillus thuringiensis
Bacillus thuringiensis is an entomopathogen in the Bacillus cereus species group, and has been used as a biopesticide for over 50 years. Despite extensive use of B. thuringiensis, there remain questions over its specific ecology compared to other members of the B. cereus group which poses problems for its continued applied use. Tying entomopathogenic ecology to a specific clade within the B. cereus group will limit confusion between B. thuringiensis used in agriculture and more harmful strains. Better understanding of B. thuringiensis ecology can also be used to combat resistance in pest species through selective passaging.
The ecology of B. thuringiensis was explored through competitions in Plutella xylostella (diamondback moth) larvae, which showed clade 2 B. thuringiensis have improved fitness in insects compared to clade 1 strains. Additionally, growth rates were compared in vitro, giving different thermal profiles for the two clades. Growth media preference was assessed for B. cereus group species with all favouring protein media over soil-based ones.
Selective passaging explored the effects of relatedness and host background on virulence evolution. For relatedness, B. thuringiensis subsp. aizawai was passaged for five rounds in P. xylostella larvae with none, one or two bottlenecking events. These treatments failed to produce any increase in virulence. In the second, B. thuringiensis subsp. entomocidus was passaged either in Cry1Ac-resistant, Cry1Ac-susceptible, alternating rounds of each or coevolved P. xylostella, with all containing a mutagenesis step with ethyl methanesulfonate. Virulence increased in the resistant and coevolved treatments, confirming that resistance is best overcome by passaging in harder-to-kill hosts.
The ecological and genetic distinctiveness of clade 2 B. thuringiensis suggests the species should be reclassified to solely this clade, which will limit safety concerns. Selective passaging can improve the virulence of strains, even if the underlying interactions are unknown; it can also provide insight into virulence evolution which would be lost when improving only at the protein level.
Abstract.
Martin SH, Singh KS, Gordon IJ, Omufwoko KS, Collins S, Warren IA, Munby H, Brattström O, Traut W, Martins DJ, et al (2019). Whole-chromosome hitchhiking driven by a male-killing endosymbiont.
2018
ffrench-Constant RH (2018). Introductory Chapter: Butterfly Wing Patterns: Not Just Painting by Numbers. In (Ed) Butterfly Wing Patterns and Mimicry, Elsevier, ix-xiii.
Traut W, Ahola V, Smith DAS, Gordon IJ, Ffrench-Constant RH (2018). Karyotypes versus Genomes: the Nymphalid Butterflies Melitaea cinxia, Danaus plexippus, and D. chrysippus.
Cytogenetic and Genome Research,
153(1), 46-53.
Abstract:
Karyotypes versus Genomes: the Nymphalid Butterflies Melitaea cinxia, Danaus plexippus, and D. chrysippus
The number of sequenced lepidopteran genomes is increasing rapidly. However, the corresponding assemblies rarely represent whole chromosomes and generally also lack the highly repetitive W sex chromosome. Knowledge of the karyotypes can facilitate genome assembly and further our understanding of sex chromosome evolution in Lepidoptera. Here, we describe the karyotypes of the Glanville fritillary Melitaea cinxia (n = 31), the monarch Danaus plexippus (n = 30), and the African queen D. chrysippus (2n = 60 or 59, depending on the source population). We show by FISH that the telomeres are of the (TTAGG) n type, as found in most insects. M. cinxia and D. plexippus have "conventional" W chromosomes which are heterochromatic in meiotic and somatic cells. In D. chrysippus, the W is inconspicuous. Neither telomeres nor W chromosomes are represented in the published genomes of M. cinxia and D. plexippus. Representation analysis in sequenced female and male D. chrysippus genomes detected an evolutionarily old autosome-Z chromosome fusion in Danaus. Conserved synteny of whole chromosomes, so called "macro synteny", in Lepidoptera permitted us to identify the chromosomes involved in this fusion. An additional and more recent sex chromosome fusion was found in D. chrysippus by karyotype analysis and classical genetics. In a hybrid population between 2 subspecies, D. c. chrysippus and D. c. dorippus, the W chromosome was fused to an autosome that carries a wing colour locus. Thus, cytogenetics and the present state of genome data complement one another to reveal the evolutionary history of the species.
Abstract.
Schaum CE, Student Research Team, Ffrench-Constant R, Lowe C, Ólafsson JS, Padfield D, Yvon-Durocher G (2018). Temperature-driven selection on metabolic traits increases the strength of an algal-grazer interaction in naturally warmed streams.
Glob Chang Biol,
24(4), 1793-1803.
Abstract:
Temperature-driven selection on metabolic traits increases the strength of an algal-grazer interaction in naturally warmed streams.
Trophic interactions are important determinants of the structure and functioning of ecosystems. Because the metabolism and consumption rates of ectotherms increase sharply with temperature, there are major concerns that global warming will increase the strength of trophic interactions, destabilizing food webs, and altering ecosystem structure and function. We used geothermally warmed streams that span an 11°C temperature gradient to investigate the interplay between temperature-driven selection on traits related to metabolism and resource acquisition, and the interaction strength between the keystone gastropod grazer, Radix balthica, and a common algal resource. Populations from a warm stream (~28°C) had higher maximal metabolic rates and optimal temperatures than their counterparts from a cold stream (~17°C). We found that metabolic rates of the population originating from the warmer stream were higher across all measurement temperatures. A reciprocal transplant experiment demonstrated that the interaction strengths between the grazer and its algal resource were highest for both populations when transplanted into the warm stream. In line with the thermal dependence of respiration, interaction strengths involving grazers from the warm stream were always higher than those with grazers from the cold stream. These results imply that increases in metabolism and resource consumption mediated by the direct, thermodynamic effects of higher temperatures on physiological rates are not mitigated by metabolic compensation in the long term, and suggest that warming could increase the strength of algal-grazer interactions with likely knock-on effects for the biodiversity and productivity of aquatic ecosystems.
Abstract.
Author URL.
2017
ffrench-Constant RH, Bass C (2017). Does resistance really carry a fitness cost?.
Current Opinion in Insect Science,
21, 39-46.
Abstract:
Does resistance really carry a fitness cost?
Insecticide resistance mutations are widely assumed to carry fitness costs. However studies to measure such costs are rarely performed on genetically related strains and are often only done in the laboratory. Theory also suggests that once evolved the cost of resistance can be offset by the evolution of fitness modifiers. But for insecticide resistance only one such example is well documented. Here we critically examine the literature on fitness costs in the absence of pesticide and ask if our knowledge of molecular biology has helped us predict the costs associated with different resistance mechanisms. We find that resistance alleles can arise from pre-existing polymorphisms and resistance associated variation can also be maintained by sexual antagonism. We describe novel mechanisms whereby both resistant and susceptible alleles can be maintained in permanent heterozygosis and discuss the likely consequences for fitness both in the presence and absence of pesticide. Taken together these findings suggest that we cannot assume that resistance always appears de novo and that our assumptions about the associated fitness costs need to be informed by a deeper understanding of the underlying molecular biology.
Abstract.
ffrench-Constant R, Waterfield N, Daborn P (2017). Insecticidal Toxins from Photorhabdus and Xenorhabdus ☆. In (Ed) Reference Module in Life Sciences, Elsevier, 704-715.
Schaum C-E, Students B, ffrench-Constant R, Lowe C, Ólafsson JS, Padfield D, Yvon-Durocher G (2017). Temperature-driven selection on metabolic traits increases the strength of an algal-grazer interaction in naturally warmed streams.
2016
Smith DAS, Gordon IJ, Traut W, Herren J, Collins S, Martins DJ, Saitoti K, Ireri P, Ffrench-Constant R (2016). A neo-W chromosome in a tropical butterfly links colour pattern, male-killing, and speciation.
Proceedings of the Royal Society B: Biological Sciences,
283(1835).
Abstract:
A neo-W chromosome in a tropical butterfly links colour pattern, male-killing, and speciation
Sexually antagonistic selection can drive both the evolution of sex chromosomes and speciation itself. The tropical butterfly the African Queen, Danaus chrysippus, shows two such sexually antagonistic phenotypes, the first being sex-linked colour pattern, the second, susceptibility to a male-killing, maternally inherited mollicute, Spiroplasma ixodeti, which causes approximately 100% mortality in male eggs and first instar larvae. Importantly, this mortality is not affected by the infection status of the male parent and the horizontal transmission of Spiroplasma is unknown. In East Africa, male-killing of the Queen is prevalent in a narrow hybrid zone centred on Nairobi. This hybrid zone separates otherwise allopatric subspecies with different colour patterns. Here we show that a neo-W chromosome, a fusion between the W (female) chromosome and an autosome that controls both colour pattern and malekilling, links the two phenotypes thereby driving speciation across the hybrid zone. Studies of the population genetics of the neo-W around Nairobi showthat the interaction between colour pattern and male-killer susceptibility restricts gene flow between two subspecies of D. chrysippus. Our results demonstrate how a complex interplay between sex, colour pattern, malekilling, and a neo-W chromosome, has set up a genetic ‘sink’ that keeps the two subspecies apart. The association between the neo-W and male-killing thus provides a ‘smoking gun’ for an ongoing speciation process.
Abstract.
Ffrench-Constant RH (2016). Butterfly gene flow goes berserk. Genome Biology, 17(1).
Ffrench-Constant RH, Williamson MS, Davies TGE, Bass C (2016). Ion channels as insecticide targets.
J Neurogenet,
30(3-4), 163-177.
Abstract:
Ion channels as insecticide targets.
Ion channels remain the primary target of most of the small molecule insecticides. This review examines how the subunit composition of heterologously expressed receptors determines their insecticide-specific pharmacology and how the pharmacology of expressed receptors differs from those found in the insect nervous system. We find that the insecticide-specific pharmacology of some receptors, like that containing subunits of the Rdl encoded GABA receptor, can be reconstituted with very few of the naturally occurring subunits expressed. In contrast, workers have struggled even to express functional insect nicotinic acetylcholine receptors (nAChRs), and work has therefore often relied upon the expression of vertebrate receptor subunits in their place. We also examine the extent to which insecticide-resistance-associated mutations, such as those in the para encoded voltage-gated sodium channel, can reveal details of insecticide-binding sites and mode of action. In particular, we examine whether mutations are present in the insecticide-binding site and/or at sites that allosterically affect the drug preferred conformation of the receptor. We also discuss the ryanodine receptor as a target for the recently developed diamides. Finally, we examine the lethality of the genes encoding these receptor subunits and discuss how this might determine the degree of conservation of the resistance-associated mutations found.
Abstract.
Author URL.
Ffrench-Constant RH, Somers-Yeates R, Bennie J, Economou T, Hodgson D, Spalding A, McGregor PK (2016). Light pollution is associated with earlier tree budburst across the United Kingdom. Proceedings of the Royal Society of London. Series B
Nadeau NJ, Pardo-Diaz C, Whibley A, Supple MA, Saenko SV, Wallbank RWR, Wu GC, Maroja L, Ferguson L, Hanly JJ, et al (2016). The gene cortex controls mimicry and crypsis in butterflies and moths.
Nature,
534(7605), 106-110.
Abstract:
The gene cortex controls mimicry and crypsis in butterflies and moths.
The wing patterns of butterflies and moths (Lepidoptera) are diverse and striking examples of evolutionary diversification by natural selection. Lepidopteran wing colour patterns are a key innovation, consisting of arrays of coloured scales. We still lack a general understanding of how these patterns are controlled and whether this control shows any commonality across the 160,000 moth and 17,000 butterfly species. Here, we use fine-scale mapping with population genomics and gene expression analyses to identify a gene, cortex, that regulates pattern switches in multiple species across the mimetic radiation in Heliconius butterflies. cortex belongs to a fast-evolving subfamily of the otherwise highly conserved fizzy family of cell-cycle regulators, suggesting that it probably regulates pigmentation patterning by regulating scale cell development. In parallel with findings in the peppered moth (Biston betularia), our results suggest that this mechanism is common within Lepidoptera and that cortex has become a major target for natural selection acting on colour and pattern variation in this group of insects.
Abstract.
Author URL.
2015
Mulley G, Beeton ML, Wilkinson P, Vlisidou I, Ockendon-Powell N, Hapeshi A, Tobias NJ, Nollmann FI, Bode HB, van den Elsen J, et al (2015). From Insect to Man: Photorhabdus Sheds Light on the Emergence of Human Pathogenicity. PLOS ONE, 10(12), e0144937-e0144937.
Ffrench-Constant RH (2015). Insect Molecular Genetics: an Introduction to Principles and Applications. Third Edition. By Marjorie A. Hoy. Academic Press. Amsterdam (The Netherlands) and Boston (Massachusetts): Elsevier. $99.95. xxvii + 808 p.; ill.; index. ISBN: 978-0-12-415874-0. 2013. The Quarterly Review of Biology, 90(1), 98-98.
Nadeau NJ, Pardo-Diaz C, Whibley A, Supple M, Wallbank R, Wu GC, Maroja L, Ferguson L, Hines H, Salazar C, et al (2015). The origins of a novel butterfly wing patterning gene from within a family of conserved cell cycle regulators.
Pauchet Y, Wielsch N, Wilkinson PA, Sakaluk SK, Svatoš A, ffrench-Constant RH, Hunt J, Heckel DG (2015). What's in the Gift? Towards a Molecular Dissection of Nuptial Feeding in a Cricket.
PLoS One,
10(10).
Abstract:
What's in the Gift? Towards a Molecular Dissection of Nuptial Feeding in a Cricket.
Nuptial gifts produced by males and transferred to females during copulation are common in insects. Yet, their precise composition and subsequent physiological effects on the female recipient remain unresolved. Male decorated crickets Gryllodes sigillatus transfer a spermatophore to the female during copulation that is composed of an edible gift, the spermatophylax, and the ampulla that contains the ejaculate. After transfer of the spermatophore, the female detaches the spermatophylax and starts to eat it while sperm from the ampulla are evacuated into the female reproductive tract. When the female has finished consuming the spermatophylax, she detaches the ampulla and terminates sperm transfer. Hence, one simple function of the spermatophylax is to ensure complete sperm transfer by distracting the female from prematurely removing the ampulla. However, the majority of orally active components of the spermatophylax itself and their subsequent effects on female behavior have not been identified. Here, we report the first analysis of the proteome of the G. sigillatus spermatophylax and the transcriptome of the male accessory glands that make these proteins. The accessory gland transcriptome was assembled into 17,691 transcripts whilst about 30 proteins were detected within the mature spermatophylax itself. of these 30 proteins, 18 were encoded by accessory gland encoded messages. Most spermatophylax proteins show no similarity to proteins with known biological functions and are therefore largely novel. A spermatophylax protein shows similarity to protease inhibitors suggesting that it may protect the biologically active components from digestion within the gut of the female recipient. Another protein shares similarity with previously characterized insect polypeptide growth factors suggesting that it may play a role in altering female reproductive physiology concurrent with fertilization. Characterization of the spermatophylax proteome provides the first step in identifying the genes encoding these proteins in males and in understanding their biological functions in the female recipient.
Abstract.
Author URL.
Ffrench-Constant RH (2015). White butterflies as solar photovoltaic concentrators. Scientific Reports, 5
2014
Ffrench-Constant RH, Dowling AJ (2014). <i>Photorhabdus</i> Toxins.
INSECT MIDGUT AND INSECTICIDAL PROTEINS,
47, 343-388.
Author URL.
Proschak A, Zhou Q, Schöner T, Thanwisai A, Kresovic D, Dowling A, ffrench‐Constant R, Proschak E, Bode HB (2014). Biosynthesis of the Insecticidal Xenocyloins in <i>Xenorhabdus bovienii</i>.
ChemBioChem,
15(3), 369-372.
Abstract:
Biosynthesis of the Insecticidal Xenocyloins in Xenorhabdus bovienii
AbstractThe biosynthesis gene cluster for the production of xenocyloins was identified in the entomopathogenic bacterium Xenorhabdus bovienii SS‐2004, and their biosynthesis was elucidated by heterologous expression and in vitro characterization of the enzymes. XclA is an S‐selective ThDP‐dependent acyloin‐like condensation enzyme, and XclB and XclC are examples of the still‐rare acylating ketosynthases that catalyze the acylation of the XclA‐derived initial xenocyloins with acetyl‐, propionyl‐, or malonyl‐CoA, thereby resulting in the formation of further xenocyloin derivatives. All xenocyloins were produced mainly by the more virulent primary variant of X. bovienii and showed activity against insect hemocytes thus contributing to the overall virulence of X. bovienii against insects.
Abstract.
ffrench-Constant RH, Dowling AJ (2014). Chapter Seven Photorhabdus Toxins. In (Ed) Insect Midgut and Insecticidal Proteins, Elsevier, 343-388.
Timmermans MJTN, Baxter SW, Clark R, Heckel DG, Vogel H, Collins S, Papanicolaou A, Fukova I, Joron M, Thompson MJ, et al (2014). Comparative genomics of the mimicry switch in<i>Papilio dardanus</i>.
Proceedings of the Royal Society B: Biological Sciences,
281(1787), 20140465-20140465.
Abstract:
Comparative genomics of the mimicry switch inPapilio dardanus
The African Mocker Swallowtail,Papilio dardanus, is a textbook example in evolutionary genetics. Classical breeding experiments have shown that wing pattern variation in this polymorphic Batesian mimic is determined by the polyallelicHlocus that controls a set of distinct mimetic phenotypes. Using bacterial artificial chromosome (BAC) sequencing, recombination analyses and comparative genomics, we show thatHco-segregates with an interval of less than 500 kb that is collinear with two other Lepidoptera genomes and contains 24 genes, including the transcription factor genesengrailed(en) andinvected(inv).His located in a region of conserved gene order, which argues against any role for genomic translocations in the evolution of a hypothesized multi-gene mimicry locus. Natural populations ofP. dardanusshow significant associations of specific morphs with single nucleotide polymorphisms (SNPs), centred onen. In addition, SNP variation in theHregion reveals evidence of non-neutral molecular evolution in theengene alone. We find evidence for a duplication potentially driving physical constraints on recombination in thelambornimorph. Absence of perfect linkage disequilibrium between different genes in the other morphs suggests thatHis limited to nucleotide positions in the regulatory and coding regions ofen. Our results therefore support the hypothesis that a single gene underlies wing pattern variation inP. dardanus.
Abstract.
Ffrench-constant R, Valencia A, Eyun SI, Wang H, Pauchet Y, Benson AK (2014). Correction: Molecular evolution of glycoside hydrolase genes in the western corn rootworm (Diabrotica virgifera virgifera) (PLoS ONE (2014) 9, 4 (e94052) DOI: 10.1371/journal.pone.0094052). PLoS ONE, 9(7).
Ffrench-Constant RH (2014). GENOMICS of monarchs and migration.
NATURE,
514(7522), 314-315.
Author URL.
Kirsch R, Gramzow L, Theißen G, Siegfried BD, ffrench-Constant RH, Heckel DG, Pauchet Y (2014). Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: Key events in the evolution of herbivory in beetles. Insect Biochemistry and Molecular Biology, 52, 33-50.
ffrench-Constant RH (2014). Insecticide resistance comes of age. Genome Biology, 15(2), 106-106.
Eyun SI, Wang H, Pauchet Y, Ffrench-Constant RH, Benson AK, Valencia-Jiménez A, Moriyama EN, Siegfried BD (2014). Molecular evolution of glycoside hydrolase genes in the western corn rootworm (Diabrotica virgifera virgifera).
PLoS ONE,
9(4).
Abstract:
Molecular evolution of glycoside hydrolase genes in the western corn rootworm (Diabrotica virgifera virgifera)
Cellulose is an important nutritional resource for a number of insect herbivores. Digestion of cellulose and other polysaccharides in plant-based diets requires several types of enzymes including a number of glycoside hydrolase (GH) families. In a previous study, we showed that a single GH45 gene is present in the midgut tissue of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). However, the presence of multiple enzymes was also suggested by the lack of a significant biological response when the expression of the gene was silenced by RNA interference. In order to clarify the repertoire of cellulose-degrading enzymes and related GH family proteins in D. v. virgifera, we performed next-generation sequencing and assembled transcriptomes from the tissue of three different developmental stages (eggs, neonates, and third instar larvae). Results of this study revealed the presence of seventy-eight genes that potentially encode GH enzymes belonging to eight families (GH45, GH48, GH28, GH16, GH31, GH27, GH5, and GH1). The numbers of GH45 and GH28 genes identified in D. v. virgifera are among the largest in insects where these genes have been identified. Three GH family genes (GH45, GH48, and GH28) are found almost exclusively in two coleopteran superfamilies (Chrysomeloidea and Curculionoidea) among insects, indicating the possibility of their acquisitions by horizontal gene transfer rather than simple vertical transmission from ancestral lineages of insects. Acquisition of GH genes by horizontal gene transfers and subsequent lineage-specific GH gene expansion appear to have played important roles for phytophagous beetles in specializing on particular groups of host plants and in the case of D. v. virgifera, its close association with maize. © 2014 Eyun et al.
Abstract.
Ffrench-Constant RH (2014). Of monarchs and migration. Nature, 514(7522), 314-315.
ffrench‐Constant RH (2014). Sex, butterflies and molecular biology: when pigmentation met mimicry. Pigment Cell & Melanoma Research, 27(4), 507-508.
2013
Swevers L, Huvenne H, Menschaert G, Kontogiannatos D, Kourti A, Pauchet Y, ffrench‐Constant R, Smagghe G (2013). <scp>C</scp>olorado potato beetle (<scp>C</scp>oleoptera) gut transcriptome analysis: expression of <scp>RNA</scp> interference‐related genes.
Insect Molecular Biology,
22(6), 668-684.
Abstract:
Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference‐related genes
AbstractIn the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double‐stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA‐mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi‐related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi‐RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.
Abstract.
Chauhan R, Jones R, Wilkinson P, Pauchet Y, Ffrench-Constant RH (2013). Cytochrome P450-encoding genes from the Heliconius genome as candidates for cyanogenesis.
Insect Molecular Biology,
22(5), 532-540.
Abstract:
Cytochrome P450-encoding genes from the Heliconius genome as candidates for cyanogenesis
Cytochrome P450s are important both in the metabolism of xenobiotics and the production of compounds such as cyanogenic glucosides, which insects use in their defence. In the present study, we use transcriptomic and genomic information to isolate and name P450-encoding genes from the butterfly Heliconius melpomene. We classify each of the putative genes into its appropriate superfamily and compare the distribution of P450s across sequenced insects. We also identify homologues of two P450s known to be involved in cyanogenesis in the six-spot Burnet moth, Zygaena filipendulae. Classification of Heliconius P450s should be an important step in the dissection of their role in the exploitation of their host plant, the passion vine Passiflora. © 2013 Royal Entomological Society.
Abstract.
Chauhan R, Jones R, Wilkinson P, Pauchet Y, Ffrench-Constant RH (2013). Cytochrome P450-encoding genes from the Heliconius genome as candidates for cyanogenesis.
Insect Mol Biol,
22(5), 532-540.
Abstract:
Cytochrome P450-encoding genes from the Heliconius genome as candidates for cyanogenesis.
Cytochrome P450s are important both in the metabolism of xenobiotics and the production of compounds such as cyanogenic glucosides, which insects use in their defence. In the present study, we use transcriptomic and genomic information to isolate and name P450-encoding genes from the butterfly Heliconius melpomene. We classify each of the putative genes into its appropriate superfamily and compare the distribution of P450s across sequenced insects. We also identify homologues of two P450s known to be involved in cyanogenesis in the six-spot Burnet moth, Zygaena filipendulae. Classification of Heliconius
P450s should be an important step in the dissection of their role in the exploitation of their host plant, the passion vine Passiflora.
Abstract.
Author URL.
Smee MR, Pauchet Y, Wilkinson P, Wee B, Singer MC, ffrench-Constant RH, Hodgson DJ, Mikheyev AS (2013). Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers.
PLoS One,
8(1).
Abstract:
Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers.
BACKGROUND: Until recently the isolation of microsatellite markers from Lepidoptera has proved troublesome, expensive and time-consuming. Following on from a previous study of Edith's checkerspot butterfly, Euphydryas editha, we developed novel microsatellite markers for the vulnerable marsh fritillary butterfly, E. aurinia. Our goal was to optimize the process in order to reduce both time and cost relative to prevailing techniques. This was accomplished by using a combination of previously developed techniques: in silico mining of a de novo assembled transcriptome sequence, and genotyping the microsatellites found there using an economic method of fluorescently labelling primers. PRINCIPAL FINDINGS: in total, we screened nine polymorphic microsatellite markers, two of which were previously published, and seven that were isolated de novo. These markers were able to amplify across geographically isolated populations throughout Continental Europe and the UK. Significant deviations from Hardy-Weinberg equilibrium were evident in some populations, most likely due to the presence of null alleles. However, we used an F(st) outlier approach to show that these markers are likely selectively neutral. Furthermore, using a set of 128 individuals from 11 populations, we demonstrate consistency in population differentiation estimates with previously developed amplified fragment length polymorphism (AFLP) markers (r = 0.68, p
Abstract.
Author URL.
Reimer D, Cowles KN, Proschak A, Nollmann FI, Dowling AJ, Kaiser M, Constant RF, Goodrich-Blair H, Bode HB (2013). Rhabdopeptides as insect-specific virulence factors from entomopathogenic bacteria. ChemBioChem
Somers-Yeates R, Hodgson D, McGregor PK, Spalding A, Ffrench-Constant RH (2013). Shedding light on moths: shorter wavelengths attract noctuids more than geometrids.
Biol Lett,
9(4).
Abstract:
Shedding light on moths: shorter wavelengths attract noctuids more than geometrids.
With moth declines reported across Europe, and parallel changes in the amount and spectra of street lighting, it is important to understand exactly how artificial lights affect moth populations. We therefore compared the relative attractiveness of shorter wavelength (SW) and longer wavelength (LW) lighting to macromoths. SW light attracted significantly more individuals and species of moth, either when used alone or in competition with LW lighting. We also found striking differences in the relative attractiveness of different wavelengths to different moth groups. SW lighting attracted significantly more Noctuidae than LW, whereas both wavelengths were equally attractive to Geometridae. Understanding the extent to which different groups of moth are attracted to different wavelengths of light will be useful in determining the impact of artificial light on moth populations.
Abstract.
Author URL.
ffrench-Constant RH (2013). The Molecular Genetics of Insecticide Resistance.
Genetics,
194(4), 807-815.
Abstract:
The Molecular Genetics of Insecticide Resistance
Abstract
. The past 60 years have seen a revolution in our understanding of the molecular genetics of insecticide resistance. While at first the field was split by arguments about the relative importance of mono- vs. polygenic resistance and field- vs. laboratory-based selection, the application of molecular cloning to insecticide targets and to the metabolic enzymes that degrade insecticides before they reach those targets has brought out an exponential growth in our understanding of the mutations involved. Molecular analysis has confirmed the relative importance of single major genes in target-site resistance and has also revealed some interesting surprises about the multi-gene families, such as cytochrome P450s, involved in metabolic resistance. Identification of the mutations involved in resistance has also led to parallel advances in our understanding of the enzymes and receptors involved, often with implications for the role of these receptors in humans. This Review seeks to provide an historical perspective on the impact of molecular biology on our understanding of resistance and to begin to look forward to the likely impact of rapid advances in both sequencing and genome-wide association analysis.
Abstract.
FFRENCH‐CONSTANT RH (2013). The Zap Trap. The Sciences, 35(2), 31-35.
Ferreira PG, Patalano S, Chauhan R, Ffrench-Constant R, Gabaldón T, Guigó R, Sumner S (2013). Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes. Genome Biology, 14(2), R20-R20.
Jones RT, Poul YL, Whibley AC, Mérot C, ffrench-Constant RH, Joron M (2013). Wing Shape Variation Associated with Mimicry in Butterflies.
Evolution,
67(8), 2323-2334.
Abstract:
Wing Shape Variation Associated with Mimicry in Butterflies
Mimetic resemblance in unpalatable butterflies has been studied by evolutionary biologists for over a century, but has largely focused on the convergence in wing color patterns. In Heliconius numata, discrete color-pattern morphs closely resemble comimics in the distantly related genus Melinaea. We examine the possibility that the shape of the butterfly wing also shows adaptive convergence. First, simple measures of forewing dimensions were taken of individuals in a cross between H. numata morphs, and showed quantitative differences between two of the segregating morphs, f. elegans and f. silvana. Second, landmark-based geometric morphometric and elliptical Fourier outline analyses were used to more fully characterize these shape differences. Extension of these techniques to specimens from natural populations suggested that, although many of the coexisting morphs could not be discriminated by shape, the differences we identified between f. elegans and f. silvana hold in the wild. Interestingly, despite extensive overlap, the shape variation between these two morphs is paralleled in their respective Melinaea comimics. Our study therefore suggests that wing-shape variation is associated with mimetic resemblance, and raises the intriguing possibility that the supergene responsible for controlling the major switch in color pattern between morphs also contributes to wing shape differences in H. numata. © 2013 the Society for the Study of Evolution.
Abstract.
2012
Dasmahapatra KK, Walters JR, Briscoe AD, Davey JW, Whibley A, Nadeau NJ, Zimin AV, Hughes DST, Ferguson LC, Martin SH, et al (2012). Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature
Dasmahapatra KK, Walters JR, Briscoe AD, Davey JW, Whibley A, Nadeau NJ, Zimin AV, Salazar C, Ferguson LC, Martin SH, et al (2012). Butterfly genome reveals promiscuous exchange of mimicry adaptations among species.
Nature,
487(7405), 94-98.
Abstract:
Butterfly genome reveals promiscuous exchange of mimicry adaptations among species
The evolutionary importance of hybridization and introgression has long been debated. Hybrids are usually rare and unfit, but even infrequent hybridization can aid adaptation by transferring beneficial traits between species. Here we use genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation. We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12, 669 predicted genes, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organization has remained broadly conserved since the Cretaceous period, when butterflies split from the Bombyx (silkmoth) lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, Heliconius melpomene, Heliconius timareta and Heliconius elevatus, especially at two genomic regions that control mimicry pattern. We infer that closely related Heliconius species exchange protective colour-pattern genes promiscuously, implying that hybridization has an important role in adaptive radiation. © 2012 Macmillan Publishers Limited.
Abstract.
ffrench-Constant RH (2012). Butterfly wing colours and patterning by numbers. Heredity, 108(6), 592-593.
Jones RT, Salazar PA, ffrench-Constant RH, Jiggins CD, Joron M (2012). Evolution of a mimicry supergene from a multilocus architecture.
Proc Biol Sci,
279(1727), 316-325.
Abstract:
Evolution of a mimicry supergene from a multilocus architecture.
The origin and evolution of supergenes have long fascinated evolutionary biologists. In the polymorphic butterfly Heliconius numata, a supergene controls the switch between multiple different forms, and results in near-perfect mimicry of model species. Here, we use a morphometric analysis to quantify the variation in wing pattern observed in two broods of H. numata with different alleles at the supergene locus, 'P'. Further, we genotype the broods to associate the variation we capture with genetic differences. This allows us to begin mapping the quantitative trait loci that have minor effects on wing pattern. In addition to finding loci on novel chromosomes, our data, to our knowledge, suggest for the first time that ancestral colour-pattern loci, known to have major effects in closely related species, may contribute to the wing patterns displayed by H. numata, despite the large transfer of effects to the supergene.
Abstract.
Author URL.
Nadeau NJ, Whibley A, Jones RT, Davey JW, Dasmahapatra KK, Baxter SW, Quail MA, Joron M, Ffrench-Constant RH, Blaxter ML, et al (2012). Genomic islands of divergence in hybridizing <i>Heliconius</i> butterflies identified by large-scale targeted sequencing.
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES,
367(1587), 343-353.
Author URL.
Grundmann F, Dill V, Dowling A, Thanwisai A, Bode E, Chantratita N, ffrench-Constant R, Bode HB (2012). Identification and isolation of insecticidal oxazoles from <i>Pseudomonas</i> spp.
Beilstein Journal of Organic Chemistry,
8, 749-752.
Abstract:
Identification and isolation of insecticidal oxazoles from Pseudomonas spp.
Two new and five known oxazoles were identified from two different Pseudomonas strains in addition to the known pyrones pseudopyronine a and B. Labeling experiments confirmed their structures and gave initial evidence for a novel biosynthesis pathway of these natural oxazoles. In order to confirm their structure, they were synthesized, which also allowed tests of their bioactivity. Additionally, the bioactivities of the synthesis intermediates were also investigated revealing interesting biological activities for several compounds despite their overall simple structures.
Abstract.
Karatolos N, Williamson MS, Denholm I, Gorman K, ffrench-Constant RH, Bass C (2012). Over-expression of a cytochrome P450 is associated with resistance to pyriproxyfen in the greenhouse whitefly trialeurodes vaporariorum.
PLoS ONE,
7(2).
Abstract:
Over-expression of a cytochrome P450 is associated with resistance to pyriproxyfen in the greenhouse whitefly trialeurodes vaporariorum
Background: the juvenile hormone mimic, pyriproxyfen is a suppressor of insect embryogenesis and development, and is effective at controlling pests such as the greenhouse whitefly Trialeurodes vaporariorum (Westwood) which are resistant to other chemical classes of insecticides. Although there are reports of insects evolving resistance to pyriproxyfen, the underlying resistance mechanism(s) are poorly understood. Results: Bioassays against eggs of a German (TV8) population of T. vaporariorum revealed a moderate level (21-fold) of resistance to pyriproxyfen. This is the first time that pyriproxyfen resistance has been confirmed in this species. Sequential selection of TV8 rapidly generated a strain (TV8pyrsel) displaying a much higher resistance ratio (>4000-fold). The enzyme inhibitor piperonyl butoxide (PBO) suppressed this increased resistance, indicating that it was primarily mediated via metabolic detoxification. Microarray analysis identified a number of significantly over-expressed genes in TV8pyrsel as candidates for a role in resistance including cytochrome-P450 dependent monooxygenases (P450s). Quantitative PCR highlighted a single P450 gene (CYP4G61) that was highly over-expressed (81.7-fold) in TV8pyrsel. Conclusion: Over-expression of a single cytochrome P450 gene (CYP4G61) has emerged as a strong candidate for causing the enhanced resistance phenotype. Further work is needed to confirm the role of the encoded P450 enzyme CYP4G61 in detoxifying pyriproxyfen. © 2012 Karatolos et al.
Abstract.
Yang G, Hernandez-Rodriguez CS, Beeton ML, Wilkinson P, Ffrench-Constant RH, Waterfield NR (2012). Pdl1 is a Putative Lipase that Enhances <i>Photorhabdus</i> Toxin Complex Secretion.
PLOS PATHOGENS,
8(5).
Author URL.
Karatolos N, Williamson MS, Denholm I, Gorman K, Ffrench-Constant R, Nauen R (2012). Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl-coenzyme a carboxylase.
Insect Molecular Biology,
21(3), 327-334.
Abstract:
Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl-coenzyme a carboxylase
Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme a carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species. © 2012 the Royal Entomological Society.
Abstract.
Nollmann FI, Dowling A, Kaiser M, Deckmann K, Grösch S, ffrench-Constant R, Bode HB (2012). Synthesis of szentiamide, a depsipeptide from entomopathogenic <i>Xenorhabdus szentirmaii</i> with activity against <i>Plasmodium falciparum</i>.
Beilstein Journal of Organic Chemistry,
8, 528-533.
Abstract:
Synthesis of szentiamide, a depsipeptide from entomopathogenic Xenorhabdus szentirmaii with activity against Plasmodium falciparum
The synthesis of the recently characterized depsipeptide szentiamide (1), which is produced by the entomopathogenic bacterium Xenorhabdus szentirmaii, is described. Whereas no biological activity was previously identified for 1, the material derived from the efficient synthesis enabled additional bioactivity tests leading to the identification of a notable activity against insect cells and Plasmodium falciparum, the causative agent of malaria.
Abstract.
Zhou Q, Dowling A, Heide H, Wöhnert J, Brandt U, Baum J, ffrench-Constant R, Bode HB (2012). Xentrivalpeptides A–Q: Depsipeptide Diversification in <i>Xenorhabdus</i>. Journal of Natural Products, 75(10), 1717-1722.
2011
Felföldi G, Eleftherianos I, Ffrench-Constant RH, Venekei I (2011). A serine proteinase homologue, SPH-3, plays a central role in insect immunity.
J Immunol,
186(8), 4828-4834.
Abstract:
A serine proteinase homologue, SPH-3, plays a central role in insect immunity.
Numerous vertebrate and invertebrate genes encode serine proteinase homologues (SPHs) similar to members of the serine proteinase family, but lacking one or more residues of the catalytic triad. These SPH proteins are thought to play a role in immunity, but their precise functions are poorly understood. In this study, we show that SPH-3 (an insect non-clip domain-containing SPH) is of central importance in the immune response of a model lepidopteran, Manduca sexta. We examine M. sexta infection with a virulent, insect-specific, Gram-negative bacterium Photorhabdus luminescens. RNA interference suppression of bacteria-induced SPH-3 synthesis severely compromises the insect's ability to defend itself against infection by preventing the transcription of multiple antimicrobial effector genes, but, surprisingly, not the transcription of immune recognition genes. Upregulation of the gene encoding prophenoloxidase and the activity of the phenoloxidase enzyme are among the antimicrobial responses that are severely attenuated on SPH-3 knockdown. These findings suggest the existence of two largely independent signaling pathways controlling immune recognition by the fat body, one governing effector gene transcription, and the other regulating genes encoding pattern recognition proteins.
Abstract.
Author URL.
Smee M, Smyth W, Tunmore M, ffrench-Constant R, Hodgson D (2011). Butterflies on the brink: Habitat requirements for declining populations of the marsh fritillary (Euphydryas aurinia) in SW England.
Journal of Insect Conservation,
15(1), 153-163.
Abstract:
Butterflies on the brink: Habitat requirements for declining populations of the marsh fritillary (Euphydryas aurinia) in SW England
1. The marsh fritillary Euphydryas aurinia is one of our most endangered butterflies, and the only to be protected under European legislation as well as British. It persists in fragile subpopulations threatened by habitat fragmentation and degradation. 2. A combination of swaling and cattle grazing are accepted to be best practice for managing wet, unimproved grasslands-the favoured habitat for E. aurinia in Cornwall. These two well-endorsed methods of management were used to increase and improve the quality of habitat for E. aurinia over a 5 years period, 2004-2008, at a stronghold network of habitat patches in mid Cornwall, south-west England. 3. Analyses of adult and larval densities over 5 years in fifty-four transects across nine sites found E. aurinia to favour habitat patches with higher densities of the larval food plant (Devil's-bit scabious Succisa pratensis), higher sward height in autumn, and intermediate optimum levels of stock grazing. 4. Main findings indicated most sites experienced significant declines in numbers. Unfavourable weather in the last 2 years of monitoring was likely to have had a significant impact on the response of individual subpopulations to habitat management though poor recovery rates may also reflect a time-lag in colonisation events after habitat improvement has occurred. 5. Habitat management produced an improvement, albeit an inconsistent improvement in habitat variables across patches-S. pratensis shows a clear recovery at some sites. Autumn sward height increased significantly at one site, and a quadratic relationship between stock grazing and important habitat variables has been found which will aid further improvement over all sites for the long term persistence of E. aurinia. © 2010 Springer Science+Business Media B.V.
Abstract.
Smee M, Smyth W, Tunmore M, Ffrench-Constant R, Hodgson D (2011). Butterflies on the brink: habitat requirements for declining populations of the marsh fritillary (Euphydryas aurinia) in SW England.
J INSECT CONSERV,
15(1-2), 153-163.
Abstract:
Butterflies on the brink: habitat requirements for declining populations of the marsh fritillary (Euphydryas aurinia) in SW England
1. The marsh fritillary Euphydryas aurinia is one of our most endangered butterflies, and the only to be protected under European legislation as well as British. It persists in fragile subpopulations threatened by habitat fragmentation and degradation. 2. A combination of swaling and cattle grazing are accepted to be best practice for managing wet, unimproved grasslands-the favoured habitat for E. aurinia in Cornwall. These two well-endorsed methods of management were used to increase and improve the quality of habitat for E. aurinia over a 5 years period, 2004-2008, at a stronghold network of habitat patches in mid Cornwall, south-west England. 3. Analyses of adult and larval densities over 5 years in fifty-four transects across nine sites found E. aurinia to favour habitat patches with higher densities of the larval food plant (Devil's-bit scabious Succisa pratensis), higher sward height in autumn, and intermediate optimum levels of stock grazing. 4. Main findings indicated most sites experienced significant declines in numbers. Unfavourable weather in the last 2 years of monitoring was likely to have had a significant impact on the response of individual subpopulations to habitat management though poor recovery rates may also reflect a time-lag in colonisation events after habitat improvement has occurred. 5. Habitat management produced an improvement, albeit an inconsistent improvement in habitat variables across patches-S. pratensis shows a clear recovery at some sites. Autumn sward height increased significantly at one site, and a quadratic relationship between stock grazing and important habitat variables has been found which will aid further improvement over all sites for the long term persistence of E. aurinia.
Abstract.
Orth JHC, Schorch B, Boundy S, Ffrench-Constant R, Kubick S, Aktories K (2011). Cell-free synthesis and characterization of a novel cytotoxic pierisin-like protein from the cabbage butterfly <i>Pieris rapae</i>.
TOXICON,
57(2), 199-207.
Author URL.
Orth JHC, Schorch B, Boundy S, Ffrench-Constant R, Kubick S, Aktories K (2011). Cell-free synthesis and characterization of a novel cytotoxic pierisin-like protein from the cabbage butterfly Pieris rapae. Toxicon, 57(2), 199-207.
Joron M, Frezal L, Jones RT, Chamberlain NL, Lee SF, Haag CR, Whibley A, Becuwe M, Baxter SW, Ferguson L, et al (2011). Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry.
NATURE,
477(7363), 203-U102.
Author URL.
Proschak A, Schultz K, Herrmann J, Dowling AJ, Brachmann AO, ffrench-Constant R, Mueller R, Bode HB (2011). Cytotoxic Fatty Acid Amides from <i>Xenorhabdus</i>.
CHEMBIOCHEM,
12(13), 2011-2015.
Author URL.
Smith DT, Hosken DJ, Rostant WG, Yeo M, Griffin RM, Bretman A, Price TAR, Ffrench-Constant RH, Wedell N (2011). DDT resistance, epistasis and male fitness in flies.
J Evol Biol,
24(6), 1351-1362.
Abstract:
DDT resistance, epistasis and male fitness in flies.
In Drosophila melanogaster, the DDT resistance allele (DDT-R) is beneficial in the presence of DDT. Interestingly, DDT-R also elevates female fitness in the absence of DDT and existed in populations before DDT use. However, DDT-R did not spread regardless of DDT-independent selective advantages in females. We ask whether sexual antagonism could explain why DDT-R did not spread before pesticide use. We tested pre- and post-copulatory male fitness correlates in two genetic backgrounds into which we backcrossed the DDT-R allele. We found costs to DDT-R that depended on the genetic background in which DDT-R was found and documented strong epistasis between genetic background and DDT-R that influenced male size. Although it remains unclear whether DDT-R is generally sexually antagonistic, or whether the fitness costs noted would be sufficient to retard the spread of DDT-R in the absence of DDT, general fitness advantages to DDT-R in the absence of DDT may be unlikely.
Abstract.
Author URL.
van Munster M, le Gleuher M, Pauchet Y, Augustin S, Courtin C, Amichot M, ffrench-Constant RH, Pauron D (2011). Molecular characterization of three genes encoding aminopeptidases N in the poplar leaf beetle Chrysomela tremulae. Insect Molecular Biology, 20(2), 267-278.
Karatolos N, Pauchet Y, Wilkinson P, Chauhan R, Denholm I, Gorman K, Nelson DR, Bass C, ffrench-Constant RH, Williamson MS, et al (2011). Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes.
BMC Genomics,
12Abstract:
Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes
Background: the whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics.Results: Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication). BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes.Conclusion: Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance. © 2011 Karatolos et al; licensee BioMed Central Ltd.
Abstract.
Terenius O, Papanicolaou A, Garbutt JS, Eleftherianos I, Huvenne H, Kanginakudru S, Albrechtsen M, an C, Aymeric J-L, Barthel A, et al (2011). RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.
JOURNAL OF INSECT PHYSIOLOGY,
57(2), 231-245.
Author URL.
2010
Jones RT, Sanchez-Contreras M, Vlisidou I, Amos MR, Yang G, Munoz-Berbel X, Upadhyay A, Potter UJ, Joyce SA, Ciche TA, et al (2010). <i>Photorhabdus</i> adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.
BMC MICROBIOLOGY,
10 Author URL.
Peat SM, ffrench-Constant RH, Waterfield NR, Marokházi J, Fodor A, Adams BJ (2010). A robust phylogenetic framework for the bacterial genus Photorhabdus and its use in studying the evolution and maintenance of bioluminescence: a case for 16S, gyrB, and glnA. Molecular Phylogenetics and Evolution, 57(2), 728-740.
Smee M, Smyth W, Tunmore M, ffrench-Constant R, Hodgson D (2010). Butterflies on the brink: habitat requirements for declining populations of the marsh fritillary (Euphydryas aurinia) in SW England. In (Ed) Lepidoptera Conservation in a Changing World, Springer Netherlands, 189-199.
Ferguson L, Lee SF, Chamberlain N, Nadeau N, Joron M, Baxter S, Wilkinson P, Papanicolaou A, Kumar S, Kee T-J, et al (2010). Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locus.
Mol Ecol,
19 Suppl 1, 240-254.
Abstract:
Characterization of a hotspot for mimicry: assembly of a butterfly wing transcriptome to genomic sequence at the HmYb/Sb locus.
The mimetic wing patterns of Heliconius butterflies are an excellent example of both adaptive radiation and convergent evolution. Alleles at the HmYb and HmSb loci control the presence/absence of hindwing bar and hindwing margin phenotypes respectively between divergent races of Heliconius melpomene, and also between sister species. Here, we used fine-scale linkage mapping to identify and sequence a BAC tilepath across the HmYb/Sb loci. We also generated transcriptome sequence data for two wing pattern forms of H. melpomene that differed in HmYb/Sb alleles using 454 sequencing technology. Custom scripts were used to process the sequence traces and generate transcriptome assemblies. Genomic sequence for the HmYb/Sb candidate region was annotated both using the MAKER pipeline and manually using transcriptome sequence reads. In total, 28 genes were identified in the HmYb/Sb candidate region, six of which have alternative splice forms. None of these are orthologues of genes previously identified as being expressed in butterfly wing pattern development, implying previously undescribed molecular mechanisms of pattern determination on Heliconius wings. The use of next-generation sequencing has therefore facilitated DNA annotation of a poorly characterized genome, and generated hypotheses regarding the identity of wing pattern at the HmYb/Sb loci.
Abstract.
Author URL.
Eleftherianos I, Ffrench-Constant RH, Clarke DJ, Dowling AJ, Reynolds SE (2010). Dissecting the immune response to the entomopathogen <i>Photorhabdus</i>.
TRENDS IN MICROBIOLOGY,
18(12), 552-560.
Author URL.
Pauchet Y, Wilkinson P, Chauhan R, Ffrench-Constant RH (2010). Diversity of beetle genes encoding novel plant cell wall degrading enzymes.
PLoS ONE,
5(12).
Abstract:
Diversity of beetle genes encoding novel plant cell wall degrading enzymes
Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent "disappearance" of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology. © 2010 Pauchet et al.
Abstract.
Dowling AJ, Wilkinson PA, Holden MTG, Quail MA, Bentley SD, Reger J, Waterfield NR, Titball RW, Ffrench-Constant RH (2010). Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.
PLoS One,
5(12).
Abstract:
Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.
Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin 'tails' and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.
Abstract.
Author URL.
Counterman BA, Araujo-Perez F, Hines HM, Baxter SW, Morrison CM, Lindstrom DP, Papa R, Ferguson L, Joron M, Ffrench-Constant RH, et al (2010). Genomic Hotspots for Adaptation: the Population Genetics of Mullerian Mimicry in <i>Heliconius erato</i>.
PLOS GENETICS,
6(2).
Author URL.
Counterman BA, Araujo-Perez F, Hines HM, Baxter SW, Morrison CM, Lindstrom DP, Papa R, Ferguson L, Joron M, Ffrench-Constant RH, et al (2010). Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in Heliconius erato.
PLoS Genet,
6(2).
Abstract:
Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in Heliconius erato.
Wing pattern evolution in Heliconius butterflies provides some of the most striking examples of adaptation by natural selection. The genes controlling pattern variation are classic examples of Mendelian loci of large effect, where allelic variation causes large and discrete phenotypic changes and is responsible for both convergent and highly divergent wing pattern evolution across the genus. We characterize nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium (LD), and candidate gene expression patterns across two unlinked genomic intervals that control yellow and red wing pattern variation among mimetic forms of Heliconius erato. Despite very strong natural selection on color pattern, we see neither a strong reduction in genetic diversity nor evidence for extended LD across either patterning interval. This observation highlights the extent that recombination can erase the signature of selection in natural populations and is consistent with the hypothesis that either the adaptive radiation or the alleles controlling it are quite old. However, across both patterning intervals we identified SNPs clustered in several coding regions that were strongly associated with color pattern phenotype. Interestingly, coding regions with associated SNPs were widely separated, suggesting that color pattern alleles may be composed of multiple functional sites, conforming to previous descriptions of these loci as "supergenes." Examination of gene expression levels of genes flanking these regions in both H. erato and its co-mimic, H. melpomene, implicate a gene with high sequence similarity to a kinesin as playing a key role in modulating pattern and provides convincing evidence for parallel changes in gene regulation across co-mimetic lineages. The complex genetic architecture at these color pattern loci stands in marked contrast to the single casual mutations often identified in genetic studies of adaptation, but may be more indicative of the type of genetic changes responsible for much of the adaptive variation found in natural populations.
Abstract.
Author URL.
Baxter SW, Nadeau NJ, Maroja LS, Wilkinson P, Counterman BA, Dawson A, Beltran M, Perez-Espona S, Chamberlain N, Ferguson L, et al (2010). Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in the Heliconius melpomene clade.
PLoS Genet,
6(2).
Abstract:
Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in the Heliconius melpomene clade.
Wing patterning in Heliconius butterflies is a longstanding example of both Müllerian mimicry and phenotypic radiation under strong natural selection. The loci controlling such patterns are "hotspots" for adaptive evolution with great allelic diversity across different species in the genus. We characterise nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium, and candidate gene expression at two loci and across multiple hybrid zones in Heliconius melpomene and relatives. Alleles at HmB control the presence or absence of the red forewing band, while alleles at HmYb control the yellow hindwing bar. Across HmYb two regions, separated by approximately 100 kb, show significant genotype-by-phenotype associations that are replicated across independent hybrid zones. In contrast, at HmB a single peak of association indicates the likely position of functional sites at three genes, encoding a kinesin, a G-protein coupled receptor, and an mRNA splicing factor. At both HmYb and HmB there is evidence for enhanced linkage disequilibrium (LD) between associated sites separated by up to 14 kb, suggesting that multiple sites are under selection. However, there was no evidence for reduced variation or deviations from neutrality that might indicate a recent selective sweep, consistent with these alleles being relatively old. of the three genes showing an association with the HmB locus, the kinesin shows differences in wing disc expression between races that are replicated in the co-mimic, Heliconius erato, providing striking evidence for parallel changes in gene expression between Müllerian co-mimics. Wing patterning loci in Heliconius melpomene therefore show a haplotype structure maintained by selection, but no evidence for a recent selective sweep. The complex genetic pattern contrasts with the simple genetic basis of many adaptive traits studied previously, but may provide a better model for most adaptation in natural populations that has arisen over millions rather than tens of years.
Abstract.
Author URL.
Jones RT, Bakker SE, Stone D, Shuttleworth SN, Boundy S, McCart C, Daborn PJ, Ffrench-Constant RH, van den Elsen JMH (2010). Homology modelling of <i>Drosophila</i> cytochrome P450 enzymes associated with insecticide resistance.
PEST MANAGEMENT SCIENCE,
66(10), 1106-1115.
Author URL.
Jones RT, Bakker SE, Stone D, Shuttleworth SN, Boundy S, McCart C, Daborn PJ, ffrench-Constant RH, van den Elsen JMH (2010). Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.
Pest Manag Sci,
66(10), 1106-1115.
Abstract:
Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.
BACKGROUND: Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. RESULTS: Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. CONCLUSION: the CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.
Abstract.
Author URL.
Wilkinson P, Paszkiewicz K, Moorhouse A, Szubert JM, Beatson S, Gerrard J, Waterfield NR, Ffrench-Constant RH (2010). New plasmids and putative virulence factors from the draft genome of an Australian clinical isolate of Photorhabdus asymbiotica.
FEMS Microbiol Lett,
309(2), 136-143.
Abstract:
New plasmids and putative virulence factors from the draft genome of an Australian clinical isolate of Photorhabdus asymbiotica.
Clinical isolates of Photorhabdus asymbiotica have been recovered from patients in both the United States of America and Australia, and the full sequence of P. asymbiotica ATCC43949 from the United States has been reported recently. In contrast to other bacteria in the genus that only infect insects, P. asymbiotica strains are able to infect both insects and humans. Using a combination of Solexa (Illumina) and 454 Life Sciences (Roche) sequence data in different assembly pipelines, we report on a draft genome sequence of a strain of P. asymbiotica recovered from a patient from Kingscliff, Australia. The best assembly yielded an N50 scaffold size of 288 627 base pairs (bp) with >88.6% of the predicted genome covered by scaffolds over 100 000 bp. One of the central differences found between this Australian isolate and the US isolate is the presence of an additional plasmid, pPAA3. This plasmid is similar to pCRY from Yersinia pestis, the causative agent of bubonic plague, and the presence of pPAA3 may account for the increased virulence of Australian isolates both against tissue culture cells and infected patients. The genome of the Kingscliff strain also contains several genomic differences from the US isolate, whose potential significance in virulence against both humans and insects is discussed.
Abstract.
Author URL.
Jones RT, Sanchez-Contreras M, Vlisidou I, Amos MR, Yang G, Muñoz-Berbel X, Upadhyay A, Potter UJ, Joyce SA, Ciche TA, et al (2010). Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.
BMC Microbiol,
10Abstract:
Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.
BACKGROUND: Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. RESULTS: a comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28 degrees C) and human (37 degrees C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. CONCLUSIONS: We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.
Abstract.
Author URL.
Eleftherianos I, Joyce S, Ffrench-Constant RH, Clarke DJ, Reynolds SE (2010). Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.
Parasitology,
137(11), 1695-1706.
Abstract:
Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.
Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.
Abstract.
Author URL.
Baxter S, McMillan O, Chamberlain N, ffrench-Constant R, Jiggins C (2010). Prospects for Locating Adaptive Genes in Lepidopteran Genomes. In (Ed) Molecular Biology and Genetics of the Lepidoptera, Taylor & Francis.
Pauchet Y, Wilkinson P, Vogel H, Nelson DR, Reynolds SE, Heckel DG, ffrench-Constant RH (2010). Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.
Insect Mol Biol,
19(1), 61-75.
Abstract:
Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.
The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.
Abstract.
Author URL.
Metcalfe K, Ffrench-Constant R, Gordon I (2010). Sacred sites as hotspots for biodiversity: the Three Sisters Cave complex in coastal Kenya.
ORYX,
44(1), 118-123.
Abstract:
Sacred sites as hotspots for biodiversity: the Three Sisters Cave complex in coastal Kenya
Sacred sites, particularly in forests, often form unofficial protected areas because their biodiversity is preserved and protected by the local people looking after the sites. Here, we survey the biodiversity of the Three Sisters Cave complex, a sacred site or kaya in a fragment of East African coastal forest in south-east Kenya. We show that, despite the tiny size of this non-gazetted forest reserve, it contains many of the threatened species of both flora (121 species) and fauna (46 species) representative of Kenya's coastal forest. Following the overexploitation and widespread destruction of coastal rainforests in Kenya, such sacred sites represent key biodiversity hotspots as well as forest islands in the now largely deforested coastal plain. Other non-gazetted forest sacred sites may represent undocumented sources of biodiversity that may contribute towards conservation of this threatened coastal habitat. © 2009 Fauna & Flora International.
Abstract.
Vlisidou I, Eleftherianos I, Dorus S, Yang G, ffrench-Constant RH, Reynolds SE, Waterfield NR (2010). The KdpD/KdpE two-component system of Photorhabdus asymbiotica promotes bacterial survival within M. sexta hemocytes.
J Invertebr Pathol,
105(3), 352-362.
Abstract:
The KdpD/KdpE two-component system of Photorhabdus asymbiotica promotes bacterial survival within M. sexta hemocytes.
Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils.
Abstract.
Author URL.
2009
ffrench-Constant RH, Steichen JC, Brun LO (2009). A molecular diagnostic for endosulfan insecticide resistance in the coffee berry borer Hypothenemus hampei (Coleoptera: Scolytidae). Bulletin of Entomological Research, 84(1), 11-15.
Eleftherianos I, Waterfield NR, Bone P, Boundy S, ffrench-Constant RH, Reynolds SE (2009). A single locus from the entomopathogenic bacterium Photorhabdus luminescens inhibits activated Manduca sexta phenoloxidase.
FEMS Microbiol Lett,
293(2), 170-176.
Abstract:
A single locus from the entomopathogenic bacterium Photorhabdus luminescens inhibits activated Manduca sexta phenoloxidase.
Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo. Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli. The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.
Abstract.
Author URL.
Zefania S, ffrench-Constant R, Long PR, Székely T (2009). Breeding distribution and ecology of the threatened Madagascar Plover Charadrius thoracicus. Ostrich, 79(1), 43-51.
Wilkinson P, Waterfield NR, Crossman L, Corton C, Sanchez-Contreras M, Vlisidou I, Barron A, Bignell A, Clark L, Ormond D, et al (2009). Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens.
BMC Genomics,
10Abstract:
Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens.
BACKGROUND: the Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago. RESULTS: We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects. CONCLUSION: Our results suggest that North American isolates of Pa have acquired virulence against man via the acquisition of a plasmid and specific virulence factors with similarity to those shown to play roles in pathogenicity against humans in other bacteria.
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Author URL.
Devonshire AL, Moores GD, Ffrench-Constant RH (2009). Detection of insecticide resistance by immunological estimation of carboxylesterase activity in Myzus persicae (Sulzer) and cross reaction of the antiserum with Phorodon humuli (Schrank) (Hemiptera: Aphididae). Bulletin of Entomological Research, 76(1), 97-107.
Ffrench-Constant RH, Devonshire AL, Clark SJ (2009). Differential rate of selection for resistance by carbamate, organophosphorus and combined pyrethroid and organophosphorus insecticides in Myzus persicae (sulzer) (Hemiptera: Aphididae). Bulletin of Entomological Research, 77(2), 227-238.
Vlisidou I, Dowling AJ, Evans IR, Waterfield N, ffrench-Constant RH, Wood W (2009). Drosophila embryos as model systems for monitoring bacterial infection in real time.
PLoS Pathog,
5(7).
Abstract:
Drosophila embryos as model systems for monitoring bacterial infection in real time.
Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid 'freezing' phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.
Abstract.
Author URL.
Ffrench-Constant RH, Clark SJ, Devonshire AL (2009). Effect of decline of insecticide residues on selection for insecticide resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae). Bulletin of Entomological Research, 78(1), 19-29.
Eleftherianos I, Felföldi G, Ffrench‐Constant RH, Reynolds SE (2009). Induced nitric oxide synthesis in the gut of <i>Manduca sexta</i> protects against oral infection by the bacterial pathogen <i>Photorhabdus luminescens</i>.
Insect Molecular Biology,
18(4), 507-516.
Abstract:
Induced nitric oxide synthesis in the gut of Manduca sexta protects against oral infection by the bacterial pathogen Photorhabdus luminescens
AbstractInjecting the insect pathogenic bacterium Photorhabdus luminescens into the blood system of the model lepidopteran insect Manduca sexta induces nitric oxide synthase (NOS) expression in the fat body and blood cells (haemocytes), whereas following oral ingestion of bacteria NOS expression is limited to the gut. We used RNA interference to knock‐down expression of NOS throughout the insect. Preventing NOS induction in this way adversely affected the survival of orally infected insects and caused a significant increase in the number of bacteria crossing into the haemolymph. By contrast, knock‐down of NOS had no effect on the mortality rate of insects infected with P. luminescens by injection. Pharmacological inhibition of NOS decreased both nitric oxide (NO) levels in the gut wall and survival of orally infected insects, whereas elevation of gut wall NO using an NO donor increased survival of NOS silenced caterpillars. Together, our results imply that induced synthesis of NO is important in mediating insect immune defence against the pathogen by inhibiting transfer of bacteria across the gut wall.
Abstract.
Waterfield N, ffrench-Constant R, Dowling A (2009). Insecticidal bacterial effectors from symbionts of insect-killing nematodes. In (Ed) Insect Symbiosis, Volume 2, Taylor & Francis, 211-224.
Papanicolaou A, Stierli R, Ffrench-Constant RH, Heckel DG (2009). Next generation transcriptomes for next generation genomes using est2assembly.
BMC Bioinformatics,
10Abstract:
Next generation transcriptomes for next generation genomes using est2assembly.
BACKGROUND: the decreasing costs of capillary-based Sanger sequencing and next generation technologies, such as 454 pyrosequencing, have prompted an explosion of transcriptome projects in non-model species, where even shallow sequencing of transcriptomes can now be used to examine a range of research questions. This rapid growth in data has outstripped the ability of researchers working on non-model species to analyze and mine transcriptome data efficiently. RESULTS: Here we present a semi-automated platform 'est2assembly' that processes raw sequence data from Sanger or 454 sequencing into a hybrid de-novo assembly, annotates it and produces GMOD compatible output, including a SeqFeature database suitable for GBrowse. Users are able to parameterize assembler variables, judge assembly quality and determine the optimal assembly for their specific needs. We used est2assembly to process Drosophila and Bicyclus public Sanger EST data and then compared them to published 454 data as well as eight new insect transcriptome collections. CONCLUSIONS: Analysis of such a wide variety of data allows us to understand how these new technologies can assist EST project design. We determine that assembler parameterization is as essential as standardized methods to judge the output of ESTs projects. Further, even shallow sequencing using 454 produces sufficient data to be of wide use to the community. est2assembly is an important tool to assist manual curation for gene models, an important resource in their own right but especially for species which are due to acquire a genome project using Next Generation Sequencing.
Abstract.
Author URL.
Aronstein K, Ode P, ffrench-Constant RH (2009). PCR based monitoring of specific Drosophila (Diptera: Drosophilidae) cyclodiene resistance alleles in the presence and absence of selection. Bulletin of Entomological Research, 85(1), 5-9.
Ahantarig A, Chantawat N, Waterfield NR, ffrench-Constant R, Kittayapong P (2009). PirAB toxin from Photorhabdus asymbiotica as a larvicide against dengue vectors.
Appl Environ Microbiol,
75(13), 4627-4629.
Abstract:
PirAB toxin from Photorhabdus asymbiotica as a larvicide against dengue vectors.
We have evaluated Photorhabdus insect-related protein (Pir) from Photorhabdus asymbiotica against dengue vectors. PirAB shows larvicidal activity against both Aedes aegypti and Aedes albopictus larvae but did not affect the Mesocyclops thermocyclopoides predator. PirAB expressed the strongest toxicity compared to PirA, PirB, or the mixture of PirA plus PirB. Whether the presence of an enterobacterial repetitive intergenic consensus sequence in PirAB, but not in PirA, PirB, or the mixture of PirA plus PirB, has any impact on biological control efficacy needs further investigation.
Abstract.
Author URL.
Eleftherianos I, Xu M, Yadi H, Ffrench-Constant RH, Reynolds SE (2009). Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.
J Exp Biol,
212(Pt 12), 1840-1848.
Abstract:
Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.
Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.
Abstract.
Author URL.
Pauchet Y, Wilkinson P, van Munster M, Augustin S, Pauron D, ffrench-Constant RH (2009). Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera.
Insect Biochem Mol Biol,
39(5-6), 403-413.
Abstract:
Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera.
The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.
Abstract.
Author URL.
Waterfield NR, Sanchez-Contreras M, Eleftherianos I, Dowling A, Yang G, Wilkinson P, Parkhill J, Thomson N, Reynolds SE, Bode HB, et al (2009). Rapid Virulence Annotation (RVA): Identification of virulence factors using a bacterial genome library and multiple invertebrate hosts (vol 105, pg 15967, 2008).
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA,
106(6), 2083-2083.
Author URL.
Smith DT, Hosken DJ, Ffrench-Constant RH, Wedell N (2009). Variation in sex peptide expression in D. melanogaster.
Genet Res (Camb),
91(4), 237-242.
Abstract:
Variation in sex peptide expression in D. melanogaster.
Male Drosophila melanogaster transfers many accessory-gland proteins to females during copulation. Sex peptide (SP) is one of these and one of its main effects is to decrease female remating propensity. To date, there has been no investigation of genetic variation in SP-gene expression levels, or if such potential variation directly influences female remating behaviour. We assessed both these possibilities and found significant variation in expression levels of the SP gene across D. melanogaster isolines. A non-linear association between SP expression levels and female remating delay suggestive of disruptive selection on expression levels was also documented. Finally, while some isolines were infected with the endosymbiont Wolbachia, no association between Wolbachia and SP expression level was found.
Abstract.
Author URL.
2008
Baxter SW, Papa R, Chamberlain N, Humphray SJ, Joron M, Morrison C, ffrench-Constant RH, McMillan WO, Jiggins CD (2008). Convergent evolution in the genetic basis of Müllerian mimicry in heliconius butterflies.
Genetics,
180(3), 1567-1577.
Abstract:
Convergent evolution in the genetic basis of Müllerian mimicry in heliconius butterflies.
The neotropical butterflies Heliconius melpomene and H. erato are Müllerian mimics that display the same warningly colored wing patterns in local populations, yet pattern diversity between geographic regions. Linkage mapping has previously shown convergent red wing phenotypes in these species are controlled by loci on homologous chromosomes. Here, AFLP bulk segregant analysis using H. melpomene crosses identified genetic markers tightly linked to two red wing-patterning loci. These markers were used to screen a H. melpomene BAC library and a tile path was assembled spanning one locus completely and part of the second. Concurrently, a similar strategy was used to identify a BAC clone tightly linked to the locus controlling the mimetic red wing phenotypes in H. erato. A methionine rich storage protein (MRSP) gene was identified within this BAC clone, and comparative genetic mapping shows red wing color loci are in homologous regions of the genome of H. erato and H. melpomene. Subtle differences in these convergent phenotypes imply they evolved independently using somewhat different developmental routes, but are nonetheless regulated by the same switch locus. Genetic mapping of MRSP in a third related species, the "tiger" patterned H. numata, has no association with wing patterning and shows no evidence for genomic translocation of wing-patterning loci.
Abstract.
Author URL.
Eleftherianos I, Baldwin H, ffrench-Constant RH, Reynolds SE (2008). Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.
J Insect Physiol,
54(1), 309-318.
Abstract:
Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.
In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.
Abstract.
Author URL.
McCart C, Ffrench-Constant RH (2008). Dissecting the insecticide-resistance- associated cytochrome P450 gene Cyp6g1.
Abstract:
Dissecting the insecticide-resistance- associated cytochrome P450 gene Cyp6g1.
Abstract.
Author URL.
Long PR, Zefania S, Ffrench-Constant RH, Szekely T (2008). Estimating the population size of an endangered shorebird, the Madagascar plover, using a habitat suitability model.
ANIMAL CONSERVATION,
11(2), 118-127.
Author URL.
Papa R, Morrison CM, Walters JR, Counterman BA, Chen R, Halder G, Ferguson L, Chamberlain N, ffrench-Constant R, Kapan DD, et al (2008). Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies. BMC Genomics, 9(1), 345-345.
van Dijk RE, Komdeur J, van der Velde M, Szentirmai I, Yang X, Ffrench-Constant R, Székely T (2008). Offspring sex ratio in the sequentially polygamous Penduline Tit Remiz pendulinus. Journal of Ornithology, 149(4), 521-527.
Waterfield NR, Sanchez-Contreras M, Eleftherianos I, Dowling A, Yang G, Wilkinson P, Parkhill J, Thomson N, Reynolds SE, Bode HB, et al (2008). Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.
Proc Natl Acad Sci U S A,
105(41), 15967-15972.
Abstract:
Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.
Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.
Abstract.
Author URL.
Lindsay HA, Baines R, ffrench-Constant R, Lilley K, Jacobs HT, O'Dell KMC (2008). The Dominant Cold-Sensitive <i>Out</i>-<i>Cold</i> Mutants of <i>Drosophila melanogaster</i> Have Novel Missense Mutations in the Voltage-Gated Sodium Channel Gene <i>paralytic</i>.
GENETICS,
180(2), 873-884.
Author URL.
Hares MC, Hinchliffe SJ, Strong PCR, Eleftherianos I, Dowling AJ, Ffrench-Constant RH, Waterfield N (2008). The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.
Microbiology (Reading),
154(Pt 11), 3503-3517.
Abstract:
The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.
The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.
Abstract.
Author URL.
2007
Iatrou K, Pierre (2007). 7<sup>th</sup>International Workshop on the Molecular Biology and Genetics of the Lepidoptera. Journal of Insect Science, 7(29), 1-5.
McCart, C. Woods, D.J. Terhzaz, S. (2007). A Drosophila systems approach to xenobiotic metabolism. Physiological Genomics, 30, 223-231.
Ffrench-Constant RH, Eleftherianos I, Reynolds SE (2007). A nematode symbiont sheds light on invertebrate immunity.
Trends Parasitol,
23(11), 514-517.
Abstract:
A nematode symbiont sheds light on invertebrate immunity.
Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.
Abstract.
Author URL.
Sayed A, Nekl ER, Siqueira HAA, Wang H-C, Ffrench-Constant RH, Bagley M, Siegfried BD (2007). A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue.
Insect Mol Biol,
16(5), 591-600.
Abstract:
A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue.
A cadherin-like gene associated with larval midgut tissues was cloned from western corn rootworm (Diabrotica virgifera virgifera: Coleoptera), an economically important agricultural pest in North America and Europe and the primary target pest species for corn hybrids expressing Cry3 toxins from Bacillus thuringiensis (Bt). The full-length cDNA (5371 bp in length) encodes an open reading frame for a 1688 amino acid polypeptide. The putative protein has similar architecture to cadherin-like proteins isolated from lepidopteran midguts that have been shown to bind to Cry1 Bt toxins and have been implicated in Bt resistance. The D. v. virgifera cadherin-like gene is expressed primarily in the larval midgut and regulated during development, with high levels of expression observed in all instars and adults but not pupae. The corresponding genomic sequence spans more than 90 kb and is interspersed with 30 large introns. The genomic organization of the cadherin-like gene for this coleopteran species bears strong resemblance to lepidopteran cadherins suggesting a common molecular basis for susceptibility to Cry3 toxins in Coleoptera.
Abstract.
Author URL.
Erickson DL, Waterfield NR, Vadyvaloo V, Long D, Fischer ER, ffrench-Constant R, Hinnebusch BJ (2007). Acute oral toxicity of <i>Yersinia pseudotuberculosis</i> to fleas:: implications for the evolution of vector-borne transmission of plague.
CELLULAR MICROBIOLOGY,
9(11), 2658-2666.
Author URL.
Boundy, S. Joyce, S. Aslam, S. (2007). An antibiotic produced by an insect-pathogenic bacterium suppresses host defences through phenoloxidase inhibition. Proceedings of the National Academy of Sciences USA, 104, 2419-2424.
Bogwitz, M.R. McCart, C. Andrianopoulos, A. (2007). Cis-regulatory elements of the Accord retrotransposon result in tissue-specific expression of the Drosophila melanogaster insecticide resistance gene Cyp6g1. Genetics, 175, 1071-1077.
Chamberlain N, Baxter S, Jiggins C, Ffrench-Constant RH (2007). Dissecting butterfly wing pattern formation in Batesian and Mullerian mimicry.
JOURNAL OF INSECT SCIENCE,
7 Author URL.
Lumb, C. Boey, A. Wong, W. (2007). Evaluating the insecticide resistance potential of eight Drosophila melanogaster cytochrome P450 genes by transgenic over-expression. Insect Biochemistry and Molecular Biology, 37, 512-519.
Baxter SW, Chamberlain N, Papa R, Humphray SJ, Ffrench-Constant RH, McMillan WO, Jiggins CD (2007). Identifying DNA markers close to quantitative traits in lepidopteran genomes:: Using wing colour variation in <i>Heliconius</i> butterflies as a model.
JOURNAL OF INSECT SCIENCE,
7 Author URL.
ffrench-Constant RH, Dowling A, Waterfield NR (2007). Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture.
Toxicon,
49(4), 436-451.
Abstract:
Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture.
Most of the insecticidal toxins used in agriculture come from a single bacterium Bacillus thuringiensis or 'Bt'. Here we review our work on the array of toxins produced by Photorhabdus and Xenorhabdus bacteria that are symbiotic with entomopathogenic nematodes, and discuss their potential for use in agriculture as alternatives to Bt. Despite the fact that both Photorhabdus and Xenorhabdus are introduced directly into the insect blood stream by their nematode vectors, they produce a range of toxins with both oral and injectable insecticidal activity. The toxin complexes (Tc's) are large orally active toxins that are displayed on the outer surface of the bacterium. They require three components (A-C) for full toxicity and one 'A' component has been successfully expressed in transgenic Arabidopsis to confer insect resistance. One such group of Tc's, the PirAB binary toxins, have oral activity against mosquitoes and some caterpillar pests. Their mode of action is not known but they show significant sequence similarity to a recently described neurotoxin beta-leptinotarsin-h isolated from the blood of the Colorado potato beetle. Other toxins such as 'makes caterpillars floppy' (Mcf) and proteins encoded by the 'Photorhabdus virulence cassettes' (PVCs) only show injectable activity. Mcf1 promotes apoptosis in a wide range of cells and appears to mimic mammalian BH3 domain-only proteins in the mitochondrion whereas the mode of action of the PVCs remains undetermined. The likely biological reasons for the massive functional redundancy in Photorhabdus insecticidal toxins are discussed.
Abstract.
Author URL.
ffrench-Constant R, Dowling A, Hares MC, Yang G, Waterfield N (2007). Photorhabdus: Genomics of a Pathogen and Symbiont. In Pallen MJ, Nelson KE, Preston GM (Eds.) Bacterial Pathogenomics, Washington D.C.: ASM Press, 419-439.
Eleftherianos I, Millichap PJ, Felfoldi G, Gokcen F, Waterfield N, Clarke DJ, Ffrench-Constant RH, Reynolds SE (2007). Probing the insect immune system with the entomopathogen <i>Photorhabdus</i>.
JOURNAL OF INSECT SCIENCE,
7 Author URL.
McCart C, ffrench‐Constant R (2007). The Costs of DDT Resistance in Drosophila and Implications for Resistance Management Strategies. In (Ed) Pesticide Chemistry, Wiley, 305-312.
Dowling AJ, Waterfield NR, Hares MC, Le Goff G, Streuli CH, ffrench-Constant RH (2007). The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain.
Cell Microbiol,
9(10), 2470-2484.
Abstract:
The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain.
Photorhabdus are Gram-negative, nematode-vectored bacteria that produce toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy 1 (Mcf1), is sufficient to allow Escherichia coli to survive within, and kill, caterpillars which are otherwise able to clear E. coli infection. Mcf1 treated caterpillars show rapid loss of body turgor (the 'floppy' phenotype) and death is associated with massive apoptosis of both the midgut epithelium and insect phagocytes. Mammalian tissue culture cells treated with Mcf1 also display key features of apoptosis including zeiosis, apoptotic nuclear morphology, DNA laddering, activation of the effector caspase-3 and PARP cleavage. As Mcf1 carries a single BH3-like domain, here we investigate the hypothesis that this toxin promotes apoptosis via the mitochondrial pathway by mimicking a BH3 domain-only protein. Consistent with this hypothesis, a double mutant within the BH3-like domain causes a dramatic decline in apoptosis. Mcf1 also alters mitochondrial membrane potential and triggers the release of cytochrome c. Cells overexpressing Bcl-x(L), an anti-apoptotic Bcl-2 family member, are resistant to Mcf1-mediated apoptosis, as are cells deficient in Bax. In addition, translocation of Bax to the mitochondrion is observed in response to Mcf1 treatment. Together, these results show that Mcf1 mediates apoptosis via the mitochondrial pathway, and are consistent with the hypothesis that the BH3-like domain in Mcf1 is a functional requirement for the pro-apoptotic activity of Mcf1.
Abstract.
Author URL.
Eleftherianos I, Gökçen F, Felföldi G, Millichap PJ, Trenczek TE, ffrench-Constant RH, Reynolds SE (2007). The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta.
Cell Microbiol,
9(5), 1137-1147.
Abstract:
The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta.
Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.
Abstract.
Author URL.
Waterfield N, Hares MC, Hinchliffe S, Wren B, ffrench-Constant R (2007). The insect Toxin complex of Yersinia. Ad Exp Med Biol, 603, 247-257.
Waterfield N, Hares M, Hinchliffe S, Wren B, ffrench-Constant R (2007). The insect toxin complex of Yersinia.
Adv Exp Med Biol,
603, 247-257.
Abstract:
The insect toxin complex of Yersinia.
Many members of the Yersinia genus encode homologues of insect toxins first observed in bacteria that are insect pathogens such as Photorhabdus, Xenorhabdus and Serratia entomophila. These bacteria secrete high molecular weight insecticidal toxins comprised of multiple protein subunits, termed the Toxin Complexes or Tc's. In Photorhabdus three distinct Tc subunits are required for full oral toxicity in insects, that include the [A], [B] and [C] types, although the exact stochiometry remains unclear. The genomes of Photorhabdus strains encode multiple tc loci, although only two have been shown to exhibit oral and injectable activity against the Hawk Moth, Manduca sexta. The exact role of the remaining homologues is unclear. The availability of bacterial genome sequences has revealed the presence of tc gene homologues in many different species. In this chapter we review the tc gene homologues in Yersinia genus. We discuss what is known about the activity of the Yersinia Tc protein homologues and attempt to relate this to the evolution of the genus and of the tca gene family.
Abstract.
Author URL.
Felfoldi G, Eleftherianos I, Ffrench-Constant RH, Venekei I, Reynolds SE (2007). The role of serineprotease homolgue 3 (SPH-3) in <i>Manduca sexta</i> shown by RNA interference.
JOURNAL OF INSECT SCIENCE,
7 Author URL.
Ffrench-Constant RH (2007). Which came first: insecticides or resistance?.
Trends Genet,
23(1), 1-4.
Abstract:
Which came first: insecticides or resistance?
Mutations that confer resistance to insecticides are well documented. However, so far, we have been unable to determine whether these mutations arose before or after the introduction of insecticides. Recently, a landmark study showed that resistance to Malathion can be detected in pinned specimens of Australian sheep blowflies that were collected before the introduction of the insecticide. This finding has numerous implications for our understanding of the prevalence of resistance to new compounds. It also indicates that pre-existing resistance alleles might not carry the fitness cost that is associated with new mutations.
Abstract.
Author URL.
2006
Andreev D, Rocheleau T, Phillips TW, Beeman RW, ffrench‐Constant RH (2006). A PCR diagnostic for cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Pest Management Science, 41(4), 345-349.
Ffrench-Constant RH, Chamberlain N, Joron M, Papa R (2006). A conserved supergene locus controls colour pattern diversity in Heliconus butterflies. PLoS Biology, 4(10), 1831-1840.
Ffrench-Constant R, Waterfield N (2006). An ABC guide to the bacterial toxin complexes.
Adv Appl Microbiol,
58, 169-183.
Author URL.
KÜPPER C, HORSBURGH GJ, DAWSON DA, FFRENCH‐CONSTANT R, SZÉKELY T, BURKE T (2006). Characterization of 36 polymorphic microsatellite loci in the Kentish plover (<i>Charadrius alexandrinus</i>) including two sex‐linked loci and their amplification in four other <i>Charadrius</i> species.
Molecular Ecology Notes,
7(1), 35-39.
Abstract:
Characterization of 36 polymorphic microsatellite loci in the Kentish plover (Charadrius alexandrinus) including two sex‐linked loci and their amplification in four other Charadrius species
AbstractWe isolated 45 new Kentish plover (Charadrius alexandrinus) microsatellite loci. These were tested for polymorphism in 42 Kentish plovers breeding in the Çukurova Delta, Turkey. Thirty‐six of the 45 loci were polymorphic with observed heterozygosity varying between 0.22 and 0.93. Genotypes of individuals of known sex indicated that two loci were sex‐linked (Calex‐26 is located on the Z chromosome and Calex‐31 on the W chromosome). Additionally, we tested all loci for amplification in four other species of Charadridae (Kittlitz's plover, Madagascar plover, three‐banded plover and white‐fronted plover). On average 34 loci amplified per species (range 29–36).
Abstract.
Coustau C, ffrench‐Constant R (2006). Detection of cyclodiene insecticide resistance‐associated mutations by single‐stranded conformational polymorphism analysis. Pest Management Science, 43(4), 267-271.
Bloomquist JR, Ffrench‐Constant RH, Roush RT (2006). Excitation of central neurons by dieldrin and picrotoxinin in susceptible and resistant Drosophila melanogaster (meigen). Pest Management Science, 32(4), 463-469.
ffrench-Constant RH, Waterfield NR (2006). Ground control for insect pests.
Nat Biotechnol,
24(6), 660-661.
Author URL.
Moores GD, Ffrench‐Constant RH, Devonshire AL (2006). Immunoassay for detecting insecticide resistance in aphids. Pest Management Science, 26(3), 324-326.
Jenkins ATA, Dash H-A, Boundy S, Halliwell CM, ffrench-Constant RH (2006). Methoxy-resorufin ether as an electrochemically active biological probe for cytochrome P450 O-demethylation.
Bioelectrochemistry,
68(1), 67-71.
Abstract:
Methoxy-resorufin ether as an electrochemically active biological probe for cytochrome P450 O-demethylation.
This paper describes the utilisation of methoxy-resorufin ether as an electrochemical probe for studying cytochrome P450 CYP6G1. Methoxy-resorufin ether is well established as a versatile substrate for cytochrome P450, as its demethylated product, resorufin, is a fluorophore. We show that in addition to these established properties, methoxy-resorufin ether also exhibits reversible two electron transfer on glassy carbon and edge plane graphite electrodes. Cyclic voltammetry measurements and differential pulse voltammetry measurements show that methoxy-resorufin ether can be easily detected at low concentrations (down to 200 nM) in a conventional three electrode electrochemical cell. These properties of methoxy-resorufin ether mean that it could be used as an electrochemical probe, to follow the rate of its demethylation by CYP6G1. We show that electrochemical measurements could discriminate between the enzyme activity of protein microsomes taken from two strains of Drosophila melanogaster (fruit fly).
Abstract.
Author URL.
Gerrard JG, Joyce SA, Clarke DJ, ffrench-Constant RH, Nimmo GR, Looke DFM, Feil EJ, Pearce L, Waterfield NR (2006). Nematode symbiont for Photorhabdus asymbiotica.
Emerg Infect Dis,
12(10), 1562-1564.
Abstract:
Nematode symbiont for Photorhabdus asymbiotica.
Photorhabdus asymbiotica is an emerging bacterial pathogen that causes locally invasive soft tissue and disseminated bacteremic infections in the United States and Australia. Although the source of infection was previously unknown, we report that the bacterium is found in a symbiotic association with an insect-pathogenic soil nematode of the genus Heterorhabditis.
Abstract.
Author URL.
Yang G, Dowling AJ, Gerike U, ffrench-Constant RH, Waterfield NR (2006). Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.
J Bacteriol,
188(6), 2254-2261.
Abstract:
Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.
Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.
Abstract.
Author URL.
Ffrench‐Constant R, Aronstein K, Anthony N, Coustau C (2006). Polymerase chain reaction‐based monitoring techniques for the detection of insecticide resistance‐associated point mutations and their potential applications. Pest Management Science, 43(3), 195-200.
Eleftherianos I, Marokhazi J, Millichap PJ, Hodgkinson AJ, Sriboonlert A, ffrench-Constant RH, Reynolds SE (2006). Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.
Insect Biochem Mol Biol,
36(6), 517-525.
Abstract:
Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.
Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.
Abstract.
Author URL.
Eleftherianos I, Millichap PJ, ffrench-Constant RH, Reynolds SE (2006). RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm <i>Manduca sexta</i> causes increased susceptibility to the insect pathogen <i>Photorhabdus</i>.
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY,
30(12), 1099-1107.
Author URL.
Eleftherianos I, Millichap PJ, ffrench-Constant RH, Reynolds SE (2006). RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus.
Dev Comp Immunol,
30(12), 1099-1107.
Abstract:
RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus.
Bacterial pathogens either hide from or overcome the immune response of their hosts. Here we show that two different species of insect pathogenic bacteria, Photorhabdus luminescens TT01 and Photorhabdus asymbiotica ATCC43949, were both recognized by the immune system of their host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and peptidoglycan recognition protein. RNA interference (RNAi)-mediated inhibition of expression ("knock-down") of each of these genes at the level of both mRNA and protein was achieved through injection of double-stranded RNA (dsRNA). Knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to either species of Photorhabdus, as measured by the rate at which infected insects died. RNAi against Immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced hemolymph phenoloxidase activity. These results show not only that Photorhabdus is recognized by the Manduca sexta immune system but also that the insect's immune system plays an active, but ultimately ineffective, role in countering infection.
Abstract.
Author URL.
ffrench-Constant R, Daborn P, Feyereisen R (2006). Resistance and the jumping gene.
Bioessays,
28(1), 6-8.
Abstract:
Resistance and the jumping gene.
Transposons are well-known architects of genetic change but their role in insecticide resistance has, until recently, only been speculated upon. Transposon insertion, or transposon-mediated transposition, could alter either metabolic enzymes capable of degrading pesticides or could change the functionality of insecticide targets. The recent work of Aminetzach and coworkers suggests an exciting alternative, that transposon insertion can cause resistance by altering gene product function. This hypothesis is discussed in the light of other examples in which transposons have been implicated in insecticide resistance.
Abstract.
Author URL.
Le Goff G, Hilliou F, Siegfried BD, Boundy S, Wajnberg E, Sofer L, Audant P, Ffrench-Constant RH, Feyereisen R (2006). Xenobiotic response in <i>Drosophila melanogaster</i>:: Sex dependence of P450 and GST gene induction.
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY,
36(8), 674-682.
Author URL.
Le Goff G, Hilliou F, Siegfried BD, Boundy S, Wajnberg E, Sofer L, Audant P, ffrench-Constant RH, Feyereisen R (2006). Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction.
Insect Biochem Mol Biol,
36(8), 674-682.
Abstract:
Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction.
The effect of xenobiotics (phenobarbital and atrazine) on the expression of Drosophila melanogaster CYP genes encoding cytochromes P450, a gene family generally associated with detoxification, was analyzed by DNA microarray hybridization and verified by real-time RT-PCR in adults of both sexes. Only a small subset of the 86 CYP genes was significantly induced by the xenobiotics. Eleven CYP genes and three glutathione S-transferases (GST) genes were significantly induced by phenobarbital, seven CYP and one GST gene were induced by atrazine. Cyp6d5, Cyp6w1, Cyp12d1 and the ecdysone-inducible Cyp6a2 were induced by both chemicals. The constitutive expression of several of the inducible genes (Cyp6a2, Cyp6a8, Cyp6d5, Cyp12d1) was higher in males than in females, and the induced level similar in both sexes. Thus, the level of induction was consistently higher in females than in males. The female-specific and hormonally regulated yolk protein genes were significantly induced by phenobarbital in males and repressed by atrazine in females. Our results suggest that the numerous CYP genes of Drosophila respond selectively to xenobiotics, providing the fly with an adaptive response to chemically adverse environments. The xenobiotic inducibility of some CYP genes previously associated with insecticide resistance in laboratory-selected strains (Cyp6a2, Cyp6a8, Cyp12d1) suggests that deregulation of P450 gene expression may be a facile way to achieve resistance. Our study also suggests that xenobiotic-induced changes in P450 levels can affect insect fitness by interfering with hormonally regulated networks.
Abstract.
Author URL.
2005
ffrench-Constant RH, Waterfield N, Daborn P (2005). 5.8 Insecticidal Toxins from Photorhabdus and Xenorhabdus. In (Ed) Comprehensive Molecular Insect Science, Elsevier, 239-253.
Ffrench-Constant R, Waterfield N (2005). An ABC Guide to the Bacterial Toxin Complexes.
Adv Appl Microbiol,
58C, 169-183.
Author URL.
Buckling A, Neilson J, Lindsay J, ffrench-Constant R, Enright M, Day N, Massey RC (2005). Clonal distribution and phase-variable expression of a major histocompatibility complex analogue protein in Staphylococcus aureus.
J Bacteriol,
187(8), 2917-2919.
Abstract:
Clonal distribution and phase-variable expression of a major histocompatibility complex analogue protein in Staphylococcus aureus.
The mapW gene of Staphylococcus aureus strain N315 contains a poly(A) tract which truncates translation of the protein. This study demonstrates that mapW is an allelic variant of the map/eap genes found in other strains and that the variation in the length of this poly(A) tract suggests that it is a contingency locus.
Abstract.
Author URL.
Buckling, A. McGhee, M. ffrench-constant, R.H. (2005). DDT resistance in flies carries no cost. Current Biology, 15(15), 587-589.
McCart C, Buckling A, Ffrench-Constant RH (2005). DDT resistance in flies carries no cost.
Curr Biol,
15(15), R587-R589.
Author URL.
Siegfried BD, Waterfield N, ffrench-Constant RH (2005). Expressed sequence tags from <i>Diabrotica virgifera virgifera</i> midgut identify a coleopteran cadherin and a diversity of cathepsins.
INSECT MOLECULAR BIOLOGY,
14(2), 137-143.
Author URL.
Siegfried BD, Waterfield N, ffrench-Constant RH (2005). Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.
Insect Mol Biol,
14(2), 137-143.
Abstract:
Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.
The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.
Abstract.
Author URL.
Eleftherianos I, ffrench-Constant RH, Reynolds SE (2005). Modulating lepidopteran immunity to bacteria using pre-infection and RNA interference.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
141(3), S109-S109.
Author URL.
Waterfield N, Hares M, Yang G, Dowling A, ffrench-Constant R (2005). Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria.
Cell Microbiol,
7(3), 373-382.
Abstract:
Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria.
The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.
Abstract.
Author URL.
Ffrench-Constant RH (2005). Something old, something transgenic, or something fungal for mosquito control?.
Trends Ecol Evol,
20(11), 577-579.
Abstract:
Something old, something transgenic, or something fungal for mosquito control?
In spite of the current research emphasis on the use of transgenic mosquitoes, insecticides are still the main method for controlling malarial mosquitoes. Although pyrethroids are the compounds of choice, insecticide resistance is now threatening the effective life of these invaluable compounds. Two recent studies have re-focused interest on entomopathogenic fungi as useful alternatives to conventional insecticides, suggesting that these fungi could be used as alternative control methods, which would thus also prolong the effective lifetime of pyrethroids.
Abstract.
Author URL.
Jenkins ATA, Buckling A, McGhee M, ffrench-Constant RH (2005). Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa.
J R Soc Interface,
2(3), 255-259.
Abstract:
Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa.
Type IV pili have been shown to play a role in the early stages of bacterial biofilm formation, but not in initial bacterial attachment. Here, using the surface analytical technique, surface plasmon resonance (SPR), we follow the attachment of the bacterium Pseudomonas aeruginosa in real time. In contrast to previous studies, we show that type IV pili mutants are defective in attachment. Both mutants lacking pili (pilA), and those possessing an overabundance of pili (pilT), showed reduced SPR measured attachment compared with the wild-type PAO1 strain. Both pil mutants also showed reduced pathogenicity in a model insect host, as measured by percentage mortality after 24h. SPR revealed differences in the kinetics of attachment between pilA and pilT, differences obscured by endpoint assays using crystal violet stain. These results highlight the power of SPR in monitoring bacterial attachment in real time and also demonstrate an additional role for type IV pili beyond bacterial aggregation and micro-colony formation.
Abstract.
Author URL.
Waterfield N, Kamita SG, Hammock BD, ffrench-Constant R (2005). The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity.
FEMS Microbiol Lett,
245(1), 47-52.
Abstract:
The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity.
The genome of the insect pathogen Photorhabdus luminescens strain TT01 contains numerous genes predicting toxins and proteases. Within the P. luminescens TT01 genome, the products of two loci, plu 4093-plu 4092 and plu 4437-plu 4436, show oral insecticidal activity against both moth and mosquito larvae. The proteins encoded by these loci, here termed 'Photorhabdus insect related' (Pir) proteins a and B, show similarity both to delta-endotoxins from Bacillus thuringiensis (Bts) and a developmentally regulated protein from a beetle, Leptinotarsa decemlineata. The beetle protein has been inferred to possess juvenile hormone esterase (JHE) activity due to its developmentally regulated pattern of expression and the Photorhabdus proteins PirA and PirB have been proposed to be mimics of insect JHEs that can disrupt insect metamorphosis by metabolizing the insect growth regulator juvenile hormone (JH) [Nat. Biotechnol. 21 (2003) 1307-1313]. Here we confirm that, when injected together, PirA and PirB from two different Photorhabdus strains have insecticidal activity against caterpillars of the moth Galleria mellonella but show no oral activity against a second moth species Manduca sexta. Direct measurement of JHE activity, however, shows that the Pir proteins are not able to metabolise JH. These data show that the Pir proteins have no JHE activity, as suggested, but leave the mode of action of these interesting proteins uncertain.
Abstract.
Author URL.
NICE CC, ANTHONY N, GELEMBIUK G, RATERMAN D, FFRENCH‐CONSTANT R (2005). The history and geography of diversification within the butterfly genus <i>Lycaeides</i> in North America.
Molecular Ecology,
14(6), 1741-1754.
Abstract:
The history and geography of diversification within the butterfly genus Lycaeides in North America
AbstractThe Lycaeides butterfly species complex in North America consists of two nominal, morphologically defined species. These butterflies are ecologically diverse and appear to be distributed as a geographically complex mosaic of locally differentiated populations that may be undergoing adaptive radiation. We asked whether patterns of molecular genetic variation within the species complex are congruent with currently recognized morphological species and whether the distribution of molecular variation is consistent with the hypothesis that Pleistocene climate changes contributed to the process of differentiation within the genus. Variation in the form of the genitalia from 726 males from 59 populations clearly distinguishes both species with only six populations containing morphologically intermediate or ambiguous individuals. However, partitioning of molecular variance in a 236 bp section of the mitochondrial AT‐rich region from 628 individuals (57 populations) surveyed using single strand conformation polymorphism analysis (SSCP) indicates that only 26% of the total genetic variation is distributed along nominal species boundaries as defined by morphology. Instead, three phylogeographical groups were detected, represented by three major haplotype clades, which account for 90% of the total genetic variance. Pleistocene glaciations appear to have fostered divergence during glacial maxima, while postglacial range expansions created opportunities for gene exchange and reticulation along suture zones between geographical groups. Data presented here allow us to make inferences about the history of the species complex. However, evidence of ancestral polymorphism and reticulation limit our ability to define species boundaries based on mitochondrial DNA sequence variation.
Abstract.
2004
Daborn P, McCart C, Woods D, ffrench-Constant RH (2004). Detection of insecticide resistance-associated mutations in cat flea <i>Rdl</i> by TaqMan-allele specific amplification.
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY,
79(1), 25-30.
Author URL.
Au C, Dean P, Reynolds SE, ffrench-Constant RH (2004). Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes.
Cell Microbiol,
6(1), 89-95.
Abstract:
Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes.
Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.
Abstract.
Author URL.
Gerrard J, Waterfield N, Vohra R, ffrench-Constant R (2004). Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen. Microbes and Infection, 6(2), 229-237.
Waterfield NR, Daborn PJ, Ffrench-Constant RH (2004). Insect pathogenicity islands in the insect pathogenic bacterium <i>Photorhabdus</i>.
Author URL.
Massey RC, Buckling A, Ffrench-Constant R (2004). Interference competition and parasite virulence.
PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES,
271(1541), 785-788.
Author URL.
Massey RC, Buckling A, ffrench-Constant R (2004). Interference competition and parasite virulence.
Proc Biol Sci,
271(1541), 785-788.
Abstract:
Interference competition and parasite virulence.
Within-host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single-strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin-producing, insect-killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non-killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single-strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections.
Abstract.
Author URL.
Waterfield NR, Wren BW, Ffrench-Constant RH (2004). Invertebrates as a source of emerging human pathogens.
Nat Rev Microbiol,
2(10), 833-841.
Abstract:
Invertebrates as a source of emerging human pathogens.
Despite their importance, little is known about the origins of many emerging human pathogens. However, given the age and current predominance of invertebrates, it is likely that bacteria-invertebrate interactions are not only a present source of human pathogens but have also shaped their evolution. Pathogens of invertebrate and unicellular organisms represent an extensive reservoir of bacterial strains equipped with virulence factors that evolved to overcome the innate immune responses of their hosts. This reservoir might represent a source of new human pathogenic strains and might also foster the spread of novel virulence factors into existing human commensal or pathogenic bacteria. This article examines the available evidence for this concept by examining pairs of closely related bacteria, one of which is benign, but insect associated, and one of which is a human pathogen.
Abstract.
Author URL.
Anthony N, Gelembiuk G, Raterman D, Nice C, Ffrench-Constant R (2004). Isolation and Characterization of Microsatellite Markers from the Endangered Karner Blue Butterfly Lycaeides Melissa Samuelis (Lepidoptera). Hereditas, 134(3), 271-273.
Jenkins ATA, Ffrench-Constant R, Buckling A, Clarke DJ, Jarvis K (2004). Study of the attachment of <i>Pseudomonas aeruginosa</i> on gold and modified gold surfaces using surface plasmon resonance.
BIOTECHNOLOGY PROGRESS,
20(4), 1233-1236.
Author URL.
Jenkins ATA, ffrench-constant R, Buckling A, Clarke DJ, Jarvis K (2004). Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance.
Biotechnol Prog,
20(4), 1233-1236.
Abstract:
Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance.
This paper describes how the technique of surface plasmon resonance (SPR) can be utilized to follow (in real time) the attachment of Pseudomonas aeruginosa bacteria on bare gold and gold modified with a self-assembled monolayer (SAM) of mercaptounadecanoic acid. We show that SPR is able to discriminate between the adsorption of live versus dead (thermally shocked) bacteria. Moreover, the SPR distinguishes between the adsorption of wild-type versus mutant bacteria (single gene knockouts), the concentration of the bacterial suspension, and between bacteria adsorbing on SAM-modified and bare gold. SPR is able to measure bacterial adsorption within seconds of the bacterial suspension being introduced. Finally, a qualitative correlation between results from SPR with a crystal violet staining assay for different mutant bacteria was observed.
Abstract.
Author URL.
Ffrench-Constant RH, Daborn PJ, Le Goff G (2004). The genetics and genomics of insecticide resistance.
Trends Genet,
20(3), 163-170.
Abstract:
The genetics and genomics of insecticide resistance.
The past ten years have seen the elucidation of the molecular basis of insect resistance to many chemical insecticides. Target genes, mostly in the nervous system, have been identified and cloned from Drosophila melanogaster and resistance-associated mutations have been examined in a range of pest insects. More recently, with the advent of annotated insect genomes, resistance mediated by complex multi-gene enzyme systems such as esterases, cytochrome p450s and glutathione-S-transferases has also been elucidated. In this article, we review the impact of Drosophila genetics on the field of insect resistance and focus on the current and future impact of genomics. These studies enable us to address three fundamental questions in the evolution of resistance. How many genes are involved? How many mutations are there within these genes? How often do these mutations arise in natural populations?
Abstract.
Author URL.
Ffrench-Constant RH, Daborn PJ, Dowling AJ, Steuli CH (2004). The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells. Cellular Microbiology, 6(4), 345-353.
Dowling AJ, Daborn PJ, Waterfield NR, Wang P, Streuli CH, ffrench-Constant RH (2004). The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells.
CELLULAR MICROBIOLOGY,
6(4), 345-353.
Author URL.
Catania, F. Kauer, M.O. (2004). World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila Melanogaster. Molecular Ecology, 13(8), 2491-2504.
2003
Ffrench-Constant R, Waterfield N, Daborn P, Joyce S, Bennett H, Au C, Dowling A, Boundy S, Reynolds S, Clarke D, et al (2003). <i>Photorhabdus</i>: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiology Reviews, 26(5), 433-456.
Bowen DJ, Rocheleau TA, Grutzmacher CK, Meslet L, Valens M, Marble D, Dowling A, Ffrench-Constant R, Blight MA (2003). Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus.
Microbiology (Reading),
149(Pt 6), 1581-1591.
Abstract:
Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus.
Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease. PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors. The metalloprotease also shows a calcium- and temperature-dependent autolysis. The prtA gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. The prtA gene resides in a locus encoding both the protease ABC transporter (prtBCD) and an intervening ORF encoding a protease inhibitor (inh). PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system. Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.
Abstract.
Author URL.
Le Goff G, Boundy S, Daborn PJ, Yen JL, Sofer L, Lind R, Sabourault C, Madi-Ravazzi L, ffrench-Constant RH (2003). Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila.
Insect Biochem Mol Biol,
33(7), 701-708.
Abstract:
Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila.
Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations.
Abstract.
Author URL.
ffrench-Constant R, Waterfield N, Daborn P, Joyce S, Bennett H, Au C, Dowling A, Boundy S, Reynolds S, Clarke D, et al (2003). Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen.
FEMS Microbiol Rev,
26(5), 433-456.
Abstract:
Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen.
Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.
Abstract.
Author URL.
DeCamillis M, ffrench-Constant R (2003). Proboscipedia represses distal signaling in the embryonic gnathal limb fields of Tribolium castaneum.
Dev Genes Evol,
213(2), 55-64.
Abstract:
Proboscipedia represses distal signaling in the embryonic gnathal limb fields of Tribolium castaneum.
Orthologs of the Hox genes Sex combs reduced ( Scr) and proboscipedia ( pd) are active in the developing labial appendages of all insect species tested. The remarkable variation among insect gnathal structures, particularly in the distal podomeres, suggests two Hox genes may enhance the adaptive potential of gnathal appendage morphology. Functional studies in the fruitfly Drosophila melanogaster, the flour beetle Tribolium castaneum and the milkweed bug Oncopeltus fasciatus show that cooperation between Scr and pb has been generally conserved, but specific mechanisms have been altered during evolution. Cross-regulation of pb by Scr is evident in Drosophila and Tribolium, the more closely related of the three species, but not in Oncopeltus. In all three species, pb function is restricted to the distal podomeres, but details are only known for Drosophila and Oncopeltus, two species exhibiting specialized stylate-haustellate mouthparts. Drosophila pb is required for distal Scr expression, and to repress the appendage patterning genes dachshund and Distal-less ( Dll). Oncopeltus pb has the novel capacity to specify leg fates. Little is known about distal functions of Tribolium pb. Hypomorphic mutations of the Tribolium pb ortholog maxillopedia can be arranged in a graded phenotypic series of palp to leg transformations along both the proximodistal and dorsoventral axes. Mid-embryonic expression profiles of Tribolium pb, Scr, wingless ( wg) and Dll genes were examined in maxillopedia hypomorphic and null mutant backgrounds. Levels of pb and Scr are significantly reduced in the distal appendage field. Tribolium pb therefore positively regulates distal Scr expression, a role that it has in common with Drosophila pb. Tribolium wg is normally down-regulated in the distal domain of the embryonic gnathal appendage buds. It becomes activated distally in maxillopedia hypomorphs. Repression of wg by pb has not been reported in the labial imaginal discs of Drosophila. Alterations of Tribolium Scr and wg expression occur in Dll-expressing cells, however, unlike in Drosophila labial imaginal discs, Dll expression appears unaffected in pb hypomorphic backgrounds. We conclude that the Hox genes Sex combs reduced and proboscipedia control an appendage organizer and cell autonomous fate determination during embryonic labial palp development in Tribolium.
Abstract.
Author URL.
Waterfield N, Daborn P, Dowling A, Yang G, Hares MC, ffrench-Constant R (2003). The insecticidal toxin Makes Caterpillars floppy 2 (Mcf2) shows similarity to HrmA, a virulence protein from a plant pathogen. Fems Microbiol Letts, 229, 265-270.
Waterfield NR, Daborn PJ, Dowling AJ, Yang G, Hares M, ffrench-Constant RH (2003). The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen.
FEMS Microbiol Lett,
229(2), 265-270.
Abstract:
The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen.
The Photorhabdus luminescens W14 toxin encoding gene makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when expressed in Escherichia coli. Here we describe a homologue of mcf (renamed as mcf1), termed mcf2, discovered in the same genome. The mcf2 gene predicts another large toxin whose central domain, like Mcf1, also shows limited homology to Clostridium cytotoxin B. However, the N-terminus of Mcf2 shows significant similarity to the type-III secreted effector HrmA from the plant pathogen Pseudomonas syringae and no similarity to the N-terminus of Mcf1. HrmA is a plant avirulence gene whose transient expression in tobacco cells results in cell death. Here we show that E. coli expressing Mcf2 can, like E. coli expressing Mcf1, kill insects. Further, expression of the c-Myc tagged N-terminus of Mcf2, the region showing similarity to HrmA, results in nuclear localisation of the fusion protein and subsequent destruction of transfected mammalian cells. The Mcf1 and Mcf2 toxins therefore belong to a family of high molecular mass toxins, differing at their N-termini, which encode different effector domains.
Abstract.
Author URL.
Marokhazi J, Waterfield N, LeGoff G, Feil E, Stabler R, Hinds J, Fodor A, ffrench-Constant RH (2003). Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.
J Bacteriol,
185(15), 4648-4656.
Abstract:
Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.
Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.
Abstract.
Author URL.
2002
Ffrench-Constant RH, Bogwitz M, Daborn PJ, Yen JL (2002). A single P450 allele associated with insecticide resistance in Drosophila. Science, 297(5590), 2253-2256.
Ffrench-Constant RH, Daborn PJ, Silva CP, Waterfield N (2002). A single Photorhabdus gene, makes caterpillars floppy (mfc), allows Escherichia coli to persist within and kill insects. Proceedings of the National Academy of Sciences, 99(16), 10742-10747.
Silva CP, Waterfield NR, Daborn PJ, Dean P, Chilver T, Au CPY, Sharma S, Potter U, Reynolds SE, ffrench-Constant RH, et al (2002). Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta.
Cell Microbiol,
4(6), 329-339.
Abstract:
Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta.
Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex a (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.
Abstract.
Author URL.
Waterfield NR, Daborn PJ, ffrench-Constant RH (2002). Genomic islands in Photorhabdus.
Trends Microbiol,
10(12), 541-545.
Abstract:
Genomic islands in Photorhabdus.
Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.
Abstract.
Author URL.
Andreasen MH, Ffrench-Constant RH (2002). In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi.
Med Vet Entomol,
16(4), 452-455.
Abstract:
In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi.
We are interested in generating a Y-autosome translocation of the Resistance to dieldrin (Rdl) locus in the malaria vector mosquito Anopheles stephensi Liston (Diptera: Culicidae), for use in sterile insect release. To ensure stability of the system, a recombination suppressing inversion can also be induced which encompasses the Rdl locus. As a first step, here we report the cloning of fragments of the Rdl gene from both An. stephensi and An. gambiae Giles using degenerate primers in the polymerase chain reaction. These fragments encode the second membrane-spanning region of the gamma-aminobutyric acid receptor and show high levels of both nucleotide and predicted amino acid identity to other Rdl-like receptors. They confirm that, as in all other arthropod species examined, dieldrin resistance in An. stephensi is associated with replacement of alanine302, in this case with a serine. In situ hybridization of the Rdl probe to polytene chromosomes of An. stephensi localizes the gene to the left arm of chromosome 3 (3L) in region 45C. Rdl localization will enable us to identify chromosomal rearrangements encompassing the Rdl locus and help anchor the genome sequence of An. gambiae to the polytene map.
Abstract.
Author URL.
Nice CC, Fordyce JA, Shapiro AM, Ffrench‐Constant R (2002). Lack of evidence for reproductive isolation among ecologically specialised lycaenid butterflies.
Ecological Entomology,
27(6), 702-712.
Abstract:
Lack of evidence for reproductive isolation among ecologically specialised lycaenid butterflies
Abstract 1. The evolution of reproductive isolation between recently diverged or incipient species is a critical component of speciation and a major focus of speciation models. In phytophagous insects, host plant fidelity (the habit of mating and ovipositing on a single host species) can contribute to assortative mating and reproductive isolation between populations adapting to alternative hosts. The potential role of host plant fidelity in the evolution of reproductive isolation was examined in a pair of North American blue butterfly species, Lycaeides idas and L. melissa.2. These species are morphologically distinct and populations of each species utilise different host plants; however they share 410 bp haplotypes of the mitochondrial cytochrome oxidase subunit I (COI) gene, indicating recent divergence.3. Some populations using native hosts exhibited strong fidelity for their natal host plant over the hosts used by nearby populations. Because these butterflies mate on or near the host plant, the development of strong host fidelity may create reproductive isolation among populations on different hosts and restrict gene flow.4. Tests of population differentiation using allozyme allele frequency data did not provide convincing evidence of restricted gene flow among populations. Based on morphological differences, observed ecological specialisation, and the sharing of genetic markers, these butterflies appear to be undergoing adaptive radiation driven at least partially by host shifts. Neutral genetic markers may fail to detect the effects of very recent host shifts in these populations due to gene flow and/or the recency of divergence and shared ancestral polymorphism.
Abstract.
Sharma S, Waterfield N, Bowen D, Rocheleau T, Holland L, James R, ffrench-Constant R (2002). The lumicins: novel bacteriocins from Photorhabdus luminescens with similarity to the uropathogenic-specific protein (USP) from uropathogenic Escherichia coli.
FEMS Microbiol Lett,
214(2), 241-249.
Abstract:
The lumicins: novel bacteriocins from Photorhabdus luminescens with similarity to the uropathogenic-specific protein (USP) from uropathogenic Escherichia coli.
Bacteriocins are proteins produced by bacteria to destroy other bacteria occupying their ecological niche. Photorhabdus luminescens is an insect pathogenic bacterium carried by an entomopathogenic nematode and occupies several different niches in its life cycle. The nematode enters the insect and releases a single strain of P. luminescens. The bacteria then kill the host and the bacteria and nematodes replicate within the cadaver. Strikingly, at the end of the infection the cadaver is still occupied by a single strain of bacterium, suggesting that P. luminescens can destroy other bacteria entering, or present within, the insect. Here we describe four loci encoding 'lumicins' in P. luminescens subsp. akhurstii strain W14. The lumicins are novel bacteriocins capable of killing other strains of Photorhabdus and Escherichia coli. These loci predict killer proteins and multiple dual type immunity proteins with domains similar to pyocins and colicins. The killer proteins are chimeric in nature with multiple domains, one of which is similar to the uropathogenic-specific protein (USP) described from uropathogenic E. coli. The implications of these novel bacteriocins for the lifestyle of Photorhabdus and the potential role of USP as a bacteriocin in E. coli are discussed.
Abstract.
Author URL.
Sharma S, Waterfield N, Bowen D, Rocheleau T, Holland L, James R, ffrench-Constant R (2002). The lumicins:: novel bacteriocins from <i>Photorhabdus luminescens</i> with similarity to the uropathogenic-specific protein (USP) from uropathogenic <i>Escherichia coli</i>.
FEMS MICROBIOLOGY LETTERS,
214(2), 241-249.
Author URL.
2001
Field LM, Devonshire AL, Ffrench-Constant RH, Forde BG (2001). Changes in DNA methylation are associated with loss of insecticide resistance in the peach‐potato aphid Myzus persicae (Sulz.). FEBS Letters, 243(2), 323-327.
Daborn P, Boundy S, Yen J, Pittendrigh B, ffrench-Constant R (2001). DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid.
Mol Genet Genomics,
266(4), 556-563.
Abstract:
DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid.
Mutagenesis can be used as a means of predicting likely mechanisms of resistance to novel classes of insecticides. We used chemical mutagenesis in Drosophila to screen for mutants that had become resistant to imidacloprid, a neonicotinoid insecticide. Here we report the isolation of two new dominant imidacloprid-resistant mutants. By recombinational mapping we show that these map to the same location as Rst(2)DDT. Furthermore, we show that pre-existing Rst(2)DDT alleles in turn confer cross-resistance to imidacloprid. In order to localize the Rst(2)DDT gene more precisely, we mapped resistance to both DDT and imidacloprid with respect to P-element markers whose genomic location is known. By screening for recombinants between these P-elements and resistance we localized the gene between 48D5-6 and 48F3-6 on the polytene chromosome map. The genomic sequence in this interval shows a cluster of cytochrome P450 genes, one of which, Cyp6g1, is over-expressed in all resistant strains examined. We are now testing the hypothesis that resistance to both compounds is associated with over-expression of this P450 gene.
Abstract.
Author URL.
Daborn PJ, Waterfield N, Blight MA, Ffrench-Constant RH (2001). Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection.
J Bacteriol,
183(20), 5834-5839.
Abstract:
Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection.
During insect infection Photorhabdus luminescens emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at approximately 48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of Photorhabdus as a model system for bacterial infection.
Abstract.
Author URL.
Waterfield N, Dowling A, Sharma S, Daborn PJ, Potter U, Ffrench-Constant RH (2001). Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.
Appl Environ Microbiol,
67(11), 5017-5024.
Abstract:
Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.
Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M. sexta when expression in E. coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E. coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P. luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P. luminescens K122 and E. coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E. coli produces visible approximately 25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.
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Author URL.
Waterfield NR, Bowen DJ, Fetherston JD, Perry RD, ffrench-Constant RH (2001). The tc genes of Photorhabdus: a growing family.
Trends Microbiol,
9(4), 185-191.
Abstract:
The tc genes of Photorhabdus: a growing family.
The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.
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Author URL.
2000
Ffrench-Constant RH, Waterfield N, Burland V, Perna NT, Daborn PJ, Bowen D, Blattner FR (2000). A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.
Appl Environ Microbiol,
66(8), 3310-3329.
Abstract:
A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.
Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g. type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.
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Author URL.
Koch PB, Lorenz U, Brakefield PM, ffrench-Constant RH (2000). Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes.
Dev Genes Evol,
210(11), 536-544.
Abstract:
Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes.
Butterfly wings are colored late in development, when pigments are synthesized in specialized wing scale cells in a fixed developmental succession. In this succession, colored pigments are deposited first and the remaining areas are later melanized black or brown. Here we studied the developmental changes underlying two wing pattern mutants, firstly melanic mutants of the swallowtail Papilio glaucus, in which the yellow background is turned black, and secondly a Spotty mutant of the satyrid Bicyclus anynana, which carries two additional eyespots. Despite the very different pattern changes in these two mutants, they are both associated with changes in rates of scale development and correspondingly, the final color pattern. In the melanic swallowtail, background scales originally destined to become yellow (normally developing early and synthesizing papiliochrome) show delayed development, fail to make papiliochrome, and subsequently melanize at the same time as scales in the wild-type black pattern. In the B. anynana eyespot, scale maturation begins with the central white focus, then progresses to the surrounding gold ring and later finishes with melanization of the black center. Mutants showing additional eyespots display accelerated rates of scale development (corresponding to new eyespots) in wing cells not normally occupied by eyespots. Thus by either delaying or accelerating rates of scale development, the final color, or position, of a wing pattern element can be changed. We propose that this heterochrony of scale development is a basic mechanism of color pattern formation on which developmental mutants act to change lepidopteran color patterns.
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Author URL.
(2000). Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito <i>Aedes aegypti</i>.
FEBS Letters,
368(3), 461-465.
Abstract:
Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti
A degenerate PCR strategy was used to isolate a fragment of the acetylcholinesterase gene (Ace) homolog from Aedes aegypti and screen for a cDNA clone containing the complete open reading frame of the gene. The predicted amino acid sequence of the Aedes gene shares 64% identify with Ace from Drosophila and 87% identity with the acetylcholinesterase gene from another mosquito species Anopheles stephensi. High levels of expression of the Aedes gene were achieved by infection of Sf21 cells with a recombinant baculovirus containing the Aedes Ace cDNA. The catalytic properties and sensitivity of the recombinant enzyme to insecticide inhibition are described and discussed in relation to the role of insensitive AChE in conferring resistance to organophosphorus and carbamate insecticides.
Abstract.
Ffrench-Constant RH, Anthony N, Aronstein K, Rocheleau T, Stilwell G (2000). Cyclodiene insecticide resistance: from molecular to population genetics.
Annu Rev Entomol,
45, 449-466.
Abstract:
Cyclodiene insecticide resistance: from molecular to population genetics.
This review follows progress in the analysis of cyclodiene insecticide resistance from the initial isolation of the mutant, through cloning of the resistance gene, to an examination of the distribution of resistance alleles in natural populations. Emphasis is given to the use of a resistant Drosophila mutant as an entry point to cloning the associated gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin. Resistance is associated with replacements of a single amino acid (alanine302) in the chloride ion channel pore of the protein. Replacements of alanine302 not only directly affect the drug binding site but also allosterically destabilize the drug preferred conformation of the receptor. Resistance is thus conferred by a unique dual mechanism associated with alanine302, which is the only residue replaced in a wide range of different resistant insects. The underlying mutations appear either to have arisen once, or multiply, depending on the population biology of the pest insect. Although resistance frequencies decline in the absence of selection, resistance alleles can persist at relatively high frequency and may cause problems for compounds to which cross-resistance is observed, such as the novel fipronils.
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Author URL.
Koch PB, Behnecke B, Weigmann-Lenz M, Ffrench-Constant RH (2000). Insect pigmentation: activities of beta-alanyldopamine synthase in wing color patterns of wild-type and melanic mutant swallowtail butterfly Papilio glaucus.
Abstract:
Insect pigmentation: activities of beta-alanyldopamine synthase in wing color patterns of wild-type and melanic mutant swallowtail butterfly Papilio glaucus.
Abstract.
Author URL.
ffrench-Constant RH, Bowen DJ (2000). Novel insecticidal toxins from nematode-symbiotic bacteria.
Cell Mol Life Sci,
57(5), 828-833.
Abstract:
Novel insecticidal toxins from nematode-symbiotic bacteria.
The current strategy of using transgenic crops expressing insecticidal protein toxins is placing increasing emphasis on the discovery of novel toxins, beyond those already derived from the bacterium Bacillus thuringiensis. Here we review the cloning of four insecticidal toxin complex (tc) encoding genes from a different bacterium Photorhabdus luminescens and of similar gene sequences from Xenorhabdus nematophilus. Both these bacteria occupy the gut of entomopathogenic nematodes and are released into the insect upon invasion by the nematode. In the insect the bacteria presumably secrete these insecticidal toxins, as well as a range of other antimicrobials, to establish the insect cadaver as a monocultural breeding ground for both bacteria and nematodes. In this review, the protein biochemistry and structure of the tc encoding loci are discussed in relation to their observed toxicity and histopathology. These toxins may prove useful as alternatives to those derived from B. thuringiensis for deployment in insect-resistant transgenic plants.
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Author URL.
Martin RL, Pittendrigh B, Liu J, Reenan R, ffrench-Constant R, Hanck DA (2000). Point mutations in domain III of a Drosophila neuronal Na channel confer resistance to allethrin. Insect Biochemistry and Molecular Biology, 30(11), 1051-1059.
Coustau C, Chevillon C, ffrench-Constant R (2000). Resistance to xenobiotics and parasites: can we count the cost?. Trends in Ecology & Evolution, 15(9), 378-383.
Bowen D, Blackburn M, Rocheleau T, Grutzmacher C, ffrench-Constant RH (2000). Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes.
Insect Biochem Mol Biol,
30(1), 69-74.
Abstract:
Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes.
Photorhabdus luminescens secretes both high molecular weight insecticidal toxin complexes and also a range of extracellular proteases into culture broth. Previous studies by others have suggested that insecticidal activity of the broth is associated with these proteases. However, by gene cloning and targeted knock-out, we have previously shown that oral insecticidal activity is associated with high molecular weight 'toxin complexes' (Tc) encoded by toxin complex or tc genes. Here we further clarify this distinction by biochemically separating the protease fractions away from the oral insecticidal activity of the Tc proteins. We purified three distinct protease fractions from the broth: one consisting of a single species of 55 kDa and two of several putatively related species of approximately 40 kDa. All of these clearly separate from the oral insecticidal activity associated with the high molecular weight Tc proteins and also show no effect on insect weight gain following injection into the haemocoel. Here we examine the substrate preferences and inhibitor profiles of these protease fractions and discuss their relationship with those previously described from other P. luminescens strains and phase variants.
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Author URL.
Koch PB, Behnecke B, ffrench-Constant RH (2000). The molecular basis of melanism and mimicry in a swallowtail butterfly.
Curr Biol,
10(10), 591-594.
Abstract:
The molecular basis of melanism and mimicry in a swallowtail butterfly.
Melanism in Lepidoptera, either industrial or in mimicry, is one of the most commonly cited examples of natural selection [1] [2]. Despite extensive studies of the frequency and maintenance of melanic genes in insect populations [1] [2], there has been little work on the underlying molecular mechanisms. Nowhere is butterfly melanism more striking than in the Eastern Tiger Swallowtail (Papilio glaucus) of North America [3] [4] [5]. In this species, females can be either yellow (wild type) or black (melanic). The melanic form is a Batesian mimic of the distasteful Pipevine Swallowtail (Battus philenor), which is also black in overall color. Melanism in P. glaucus is controlled by a single Y-linked (female) black gene [6]. Melanic females, therefore, always have melanic daughters. Black melanin replaces the background yellow in melanic females. Here, we show that the key enzyme involved is N-beta-alanyl-dopamine-synthase (BAS), which shunts dopamine from the melanin pathway into the production of the yellow color pigment papiliochrome and also provides products for cuticle sclerotization. In melanic females, this enzyme is suppressed, leading to abnormal melanization of a formerly yellow area, and wing scale maturation is also delayed in the same area. This raises the possibility that either reduced BAS activity itself is preventing scale sclerotization (maturation) or, in contrast, that the delay in scale maturation precludes expression of BAS at the correct stage. Together, these data show how changes in expression of a single gene product could result in multiple wing color phenotypes. The implications for the genetic control of mimicry in other Lepidoptera are discussed.
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Author URL.
Crennell SJ, Tickler PM, Bowen DJ, ffrench-Constant RH (2000). The predicted structure of photopexin from Photorhabdus shows the first haemopexin-like motif in prokaryotes.
FEMS Microbiol Lett,
191(1), 139-144.
Abstract:
The predicted structure of photopexin from Photorhabdus shows the first haemopexin-like motif in prokaryotes.
The insect pathogenic bacterium Photorhabdus luminescens secretes several insecticidal high molecular mass 'toxin complexes'. Analysis of the putative pathogenicity island surrounding the toxin complex a (tca) locus revealed two open reading frames (ORFs) of unknown function. The predicted protein sequences of these ORFs show a repeated motif similar to those found in the vertebrate haem scavenging molecule haemopexin, limunectin (a phosphocholine binding protein from Limulus) and the C-terminal domains of matrix metalloproteinases (MMPs) (where they are thought to be important for cell attachment and adhesion). We have therefore named the operon photopexin AB and the putative encoded proteins 'photopexins' a and B (PpxA and PpxB). The predicted amino acid sequence of PpxA was modelled onto the crystal structure of a MMP. Our model predicts not only that PpxA and PpxB have beta-propeller domains but also that each haemopexin-like repeat corresponds to one blade of a propeller, suggesting the limunectin structure itself may also contain two or three such haemopexin-like propellers. The overall structure of PpxA has striking similarity to that of haemopexin suggesting that it may be used by the bacterium to scavenge iron containing compounds from insects. The implications for the potential role of Ppx proteins in pathogenicity are discussed. This is the first finding of a haemopexin-like repeat outside plants and animals.
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Author URL.
1999
Wang CT, Zhang HG, Rocheleau TA, ffrench-Constant RH, Jackson MB (1999). Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.
Biophys J,
77(2), 691-700.
Abstract:
Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.
To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.
Abstract.
Author URL.
Bowen D, Blackburn M, Rocheleau TA, Andreev O, Golubeva E, ffrench‐Constant RH (1999). Insecticidal toxins from the bacterium Photorhabdus luminescens: gene cloning and toxin histopathology. Pest Management Science, 55(6), 666-668.
ffrench-Constant RH (1999). Insecticide resistance and nucleotide variability in the coffee berry borer.
Author URL.
Andreev D, Kreitman M, Phillips TW, Beeman RW, ffrench-Constant RH (1999). Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae).
J Mol Evol,
48(5), 615-624.
Abstract:
Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae).
The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a gamma-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5-6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution.
Abstract.
Author URL.
ffrench-Constant R, Bowen D (1999). Photorhabdus toxins: novel biological insecticides.
Curr Opin Microbiol,
2(3), 284-288.
Abstract:
Photorhabdus toxins: novel biological insecticides.
Following concerns over the potential for insect resistance to insecticidal Bacillus thuringiensis toxins expressed in transgenic plants, there has been recent interest in novel biological insecticides. Over the past year there has been considerable progress in the cloning of several alternative toxin genes from the bacteria Photorhabdus luminescens and Xenorhabdus nematophilus. These genes encode large insecticidal toxin complexes with little homology to other known toxins.
Abstract.
Author URL.
ffrench-Constant RH (1999). Target site mediated insecticide resistance: what questions remain?.
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY,
29(5), 397-403.
Author URL.
ffrench-Constant RH, Pittendrigh B, Vaughan A, Anthony N (1999). Why are there so few insecticide resistance-associated mutations?.
Author URL.
1998
Blackburn M, Golubeva E, Bowen D, Ffrench-Constant RH (1998). A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta.
Appl Environ Microbiol,
64(8), 3036-3041.
Abstract:
A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta.
Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (delta-endotoxins and Vip's), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.
Abstract.
Author URL.
Liu HT, Stilwell G, Anthony N, Rocheleau T, ffrench-Constant RH (1998). Analysis of a mosquito acetylcholinesterase gene promoter.
Insect Mol Biol,
7(1), 11-17.
Abstract:
Analysis of a mosquito acetylcholinesterase gene promoter.
Insect acetylcholinesterase is the target site for organophosphorus and carbamate insecticides and point mutations in the Ace gene are associated with resistance in Drosophila melanogaster and Musca domestica. However, little is known of the genetic regulation of insect Ace genes. Here we report the isolation of four different cDNAs from an Aedes Ace locus and identification of the gene promoter. Northern analysis reveals two large (>10 kb) transcripts and one smaller transcript of 4 kb. The region containing the initiation of transcription was localized by sequencing the two 5' most cDNAs and by 5' RACE. The transcription start point was subsequently identified by primer extension and is flanked by a perfect arthropod initiator consensus sequence. The promoter lacks a TATA box but contains several matches to other consensus sequences for eukaryotic transcription factors. In common with the Drosophila Ace gene, there are also multiple potential initiators of translation (ATGs) upstream of the main open reading frame. The structure of the 5' leader and promoter is compared to that found in other insect and vertebrate Ace genes and the possibility that this locus is homologous to one of two Ace loci described in another mosquito, Culex pipiens, is discussed.
Abstract.
Author URL.
Vaughan A, Chadee DD, French-Constant R (1998). Biochemical monitoring of organophosphorus and carbamate insecticide resistance in Aedes aegypti mosquitoes from Trinidad.
Med Vet Entomol,
12(3), 318-321.
Author URL.
Anthony NM, Brown JK, Feyereisen R, ffrench-Constant RH (1998). Diagnosis and characterization of insecticide-insensitive acetylcholinesterase in three populations of the sweetpotato whitefly Bemisia tabaci.
PESTICIDE SCIENCE,
52(1), 39-46.
Author URL.
Anthony N, Unruh T, Ganser D, ffrench-Constant R (1998). Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae.
Mol Gen Genet,
260(2-3), 165-175.
Abstract:
Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae.
Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a gamma-aminobutyric acid (GABA) receptor subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Alanine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, this same alanine residue is replaced either by a serine, or, in some resistant strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of the green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other alleles with the following amino acid replacements: A302-->Glycine (allele G), A302-->SerineTCG (allele S) and A302-->SerineAGT (allele S'). Evidence from direct nucleotide sequencing and Single Stranded Conformational Polymorphism (SSCP) analysis shows that at least three of these different Rdl alleles (i.e. A, G and S) are commonly present in individual aphids or aphid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutation confirm the presence of two independent Rdl-like loci in M. persicae. One locus carries the susceptible alanine (A) and/or resistant glycine (G) allele while the other carries the two serine alleles (S or S'). Whereas resistance levels are correlated with the glycine replacement, the S allele was present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacements at the first (A/G) but not the second (S/S') locus. Phylogenetic analysis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen through a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit diversity are discussed.
Abstract.
Author URL.
Bowen D, Rocheleau TA, Blackburn M, Andreev O, Golubeva E, Bhartia R, ffrench-Constant RH (1998). Insecticidal toxins from the bacterium Photorhabdus luminescens.
Science,
280(5372), 2129-2132.
Abstract:
Insecticidal toxins from the bacterium Photorhabdus luminescens.
Transgenic plants expressing Bacillus thuringiensis (Bt) toxins are currently being deployed for insect control. In response to concerns about Bt resistance, we investigated a toxin secreted by a different bacterium Photorhabdus luminescens, which lives in the gut of entomophagous nematodes. In insects infected by the nematode, the bacteria are released into the insect hemocoel; the insect dies and the nematodes and bacteria replicate in the cadaver. The toxin consists of a series of four native complexes encoded by toxin complex loci tca, tcb, tcc, and tcd. Both tca and tcd encode complexes with high oral toxicity to Manduca sexta and therefore they represent potential alternatives to Bt for transgenic deployment.
Abstract.
Author URL.
Andreev D, Breilid H, Kirkendall L, Brun LO, ffrench-Constant RH (1998). Lack of nucleotide variability in a beetle pest with extreme inbreeding.
Insect Mol Biol,
7(2), 197-200.
Abstract:
Lack of nucleotide variability in a beetle pest with extreme inbreeding.
The coffee berry borer beetle Hypothenemus hampei (Ferrari) (Curculionidae: Scolytinae) is the major insect pest of coffee and has spread to most of the coffee-growing countries of the world. This beetle also displays an unusual life cycle, with regular sibling mating. This regular inbreeding and the population bottlenecks occurring on colonization of new regions should lead to low levels of genetic diversity. We were therefore interested in determining the level of nucleotide variation in nuclear and mitochondrial genomes of this beetle worldwide. Here we show that two nuclear loci (Resistance to dieldrin and ITS2) are completely invariant, whereas some variability is maintained at a mitochondrial locus (COI), probably corresponding to a higher mutation rate in the mitochondrial genome. Phylogenetic analysis of the mitochondrial data shows only two clades of beetle haplotypes outside of Kenya, the proposed origin of the species. These data confirm that inbreeding greatly reduces nucleotide variation and suggest the recent global spread of only two inbreeding lines of this bark beetle.
Abstract.
Author URL.
Koch PB, Keys DN, Rocheleau T, Aronstein K, Blackburn M, Carroll SB, ffrench-Constant RH (1998). Regulation of dopa decarboxylase expression during colour pattern formation in wild-type and melanic tiger swallowtail butterflies.
Development,
125(12), 2303-2313.
Abstract:
Regulation of dopa decarboxylase expression during colour pattern formation in wild-type and melanic tiger swallowtail butterflies.
The eastern tiger swallowtail butterfly Papilio glaucus shows a striking example of Batesian mimicry. In this species, females are either wild type (yellow and black) or melanic (where most of the yellow colour is replaced by black). In order to understand how these different colour patterns are regulated, we examined the temporal order of wing pigment synthesis via precursor incorporation studies, enzyme assays, and in situ hybridisation to mRNA encoding a key enzyme, dopa decarboxylase. We show that dopa decarboxylase provides dopamine to both of the two major colour pigments, papiliochrome (yellow) and melanin (black). Interestingly, however, dopa decarboxylase activity is spatially and temporally regulated, being utilised early in presumptive yellow tissues and later in black. Further, in melanic females, both dopa decarboxylase activity and early papiliochrome synthesis are suppressed in the central forewing and this normally yellow area is later melanised. These results show that the regulation of enzyme synthesis observed in the yellow/black pattern of a single wing, is similar to that involved in melanism. We infer that dopa decarboxylase activity must be regulated in concert with downstream enzymes of either the melanin and/or the papiliochrome specific pathways, forming part of a developmental switch between yellow or black. This modification of multiple enzyme activities in concert is consistent with a model of melanisation involving coordinate regulation of the underlying synthetic pathways by a single Y-linked (female) factor.
Abstract.
Author URL.
Stilwell GE, ffrench-Constant RH (1998). Transcriptional analysis of the Drosophila GABA receptor gene resistance to dieldrin.
J Neurobiol,
36(4), 468-484.
Abstract:
Transcriptional analysis of the Drosophila GABA receptor gene resistance to dieldrin.
The Resistance to dieldrin (Rdl) gene encodes a novel subunit of a gamma-aminobutyric acid (GABA)-gated chloride ion channel in Drosophila. We were interested in defining the spatial and temporal expression pattern of this gene and in understanding the basis of its regulation. Rdl is expressed in both the embryonic central and peripheral nervous system. Here, we describe the complete Rdl transcription unit (approximately 50 kb) via localization of the flanking transcripts. The Rdl transcript itself is large (8.8 kb) and is composed of a short open reading frame (2 kb) with exceptionally long 5' (1.8-kb) and 3' (5-kb) untranslated regions (UTRs). The correct spatial and temporal expression of Rdl can be rescued by transformation constructs containing only 3.5 kb of DNA, a region which encompasses the transcription start point (tsp). This region also contains sequences strikingly similar to those found in other ion channel gene promoters. Failure of minigene constructs lacking the long 3' UTR to fully rescue both the lethal and resistance phenotypes associated with the Rdl locus might arise owing to the role of these sequences in message stability or trafficking.
Abstract.
Author URL.
ffrench-Constant RH, Pittendrigh B, Vaughan A, Anthony N (1998). Why are there so few resistance-associated mutations in insecticide target genes?.
Abstract:
Why are there so few resistance-associated mutations in insecticide target genes?
Abstract.
Author URL.
1997
Pittendrigh B, Aronstein K, Zinkovsky E, Andreev O, Campbell B, Daly J, Trowell S, Ffrench-Constant RH (1997). Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.
Insect Biochem Mol Biol,
27(6), 507-512.
Abstract:
Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.
The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.
Abstract.
Author URL.
Brun LO, Ffrench-Constant RH (1997). Insecticide resistance in the coffee berry borer: State of current knowledge.
Author URL.
Hosie AM, Aronstein K, Sattelle DB, ffrench-Constant RH (1997). Molecular biology of insect neuronal GABA receptors.
Trends Neurosci,
20(12), 578-583.
Abstract:
Molecular biology of insect neuronal GABA receptors.
Ionotropic gamma-aminobutyric acid (GABA) receptors are distributed throughout the nervous systems of many insect species. As with their vertebrate counterparts, GABAA receptors and GABAC receptors, the binding of GABA to ionotropic insect receptors elicits a rapid, transient opening of anion-selective ion channels which is generally inhibitory. Although insect and vertebrate GABA receptors share a number of structural and functional similarities, their pharmacology differs in several aspects. Recent studies of cloned Drosophila melanogaster GABA receptors have clarified the contribution of particular subunits to these differences. Insect ionotropic GABA receptors are also the target of numerous insecticides and an insecticide-resistant form of a Drosophila GABA-receptor subunit has enhanced our understanding of the structure-function relationship of one aspect of pharmacology common to both insect and vertebrate GABA receptors, namely antagonism by the plant-derived toxin picrotoxinin.
Abstract.
Author URL.
Severson DW, Anthony NM, Andreev O, ffrench-Constant RH (1997). Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti).
J Hered,
88(6), 520-524.
Abstract:
Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti).
Several loci conferring insecticide resistance in the yellow fever mosquito (Aedes aegypti) have previously been mapped by simple recombinational mapping. Here we describe correlation of these resistance phenotypes with molecular gene probes for insecticide target sites by RFLP mapping. The para sodium channel gene homologue and the GABA receptor gene Resistance to dieldrin map to the same genome regions as the DDT/pyrethroid and cyclodiene resistance loci, respectively. Although the acetylcholinesterase (target site of organophosphorus and carbamate insecticides) gene Ace does not map to any known resistance locus, it maps very close to the sex-determining locus. We discuss the possibilities that, if identified, Ace-mediated resistance in A. aegypti will be sex linked or that, as suggested for anopheline mosquitoes, two independent Ace loci may exist, one of which is autosomal. These results support the importance of target site insensitivity as an insecticide resistance mechanism in mosquitoes.
Abstract.
Author URL.
Breilid H, Brun LO, Andreev D, Ffrench-Constant RH, Kirkendall LR (1997). Phylogeographic patterns of introduced populations of the coffee berry borer Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) inferred from mitochondrial DNA sequences.
Author URL.
Pittendrigh B, Reenan R, ffrench-Constant RH, Ganetzky B (1997). Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides.
Mol Gen Genet,
256(6), 602-610.
Abstract:
Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides.
The gene para in Drosophila melanogaster encodes an alpha subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of targetsite resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed.
Abstract.
Author URL.
Vaughan A, Rocheleau T, ffrench-Constant R (1997). Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.
Exp Parasitol,
87(3), 237-244.
Abstract:
Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.
Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described from Drosophila, the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti as a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.
Abstract.
Author URL.
Dunkov BC, Guzov VM, Mocelin G, Shotkoski F, Brun A, Amichot M, Ffrench-Constant RH, Feyereisen R (1997). The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital.
DNA Cell Biol,
16(11), 1345-1356.
Abstract:
The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital.
The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.
Abstract.
Author URL.
Brotz TM, Bochenek B, Aronstein K, Ffrench-Constant RH, Borst A (1997). gamma-Aminobutyric acid receptor distribution in the mushroom bodies of a fly (Calliphora erythrocephala): a functional subdivision of Kenyon cells?.
J Comp Neurol,
383(1), 42-48.
Abstract:
gamma-Aminobutyric acid receptor distribution in the mushroom bodies of a fly (Calliphora erythrocephala): a functional subdivision of Kenyon cells?
Antibodies against the Drosophila gamma-aminobutyric acid (GABA) receptor subunit RDL were used to investigate the significance of inhibitory inputs to the mushroom bodies in the blowfly (Calliphora erythrocephala) brain. The pedunculus and the lobes of the mushroom body, which mainly consist of Kenyon cell fibers, revealed strong immunoreactivity against RDL. Pedunculi, alpha- and beta-lobe show characteristic unstained core structures with concentric labeling along the neuropile axis. The gamma-lobes in contrast exhibit a compartmentalized RDL-immunoreactive pattern. These data suggest an important role of GABAergic inhibition in the pedunculus and the lobes of insect mushroom bodies. It is most likely that the RDL-immunoreactivity in the mushroom bodies is closely related to Kenyon cell fibers suggesting that Kenyon cells are an inhomogeneous class of neurons, only part of which receive inhibitory GABAergic input from extrinsic elements. GABAergic inhibition, therefore, may play a substantial role in the process of learning and memory formation in the insect mushroom bodies.
Abstract.
Author URL.
1996
Dunkov BC, Rodriguez-Arnaiz R, Pittendrigh B, ffrench-Constant RH, Feyereisen R (1996). Cytochrome P450 gene clusters in. MGG Molecular & General Genetics, 251(3), 290-290.
Dunkov BC, Rodriguez-Arnaiz R, Pittendrigh B, ffrench-Constant RH, Feyereisen R (1996). Cytochrome P450 gene clusters in Drosophila melanogaster.
Mol Gen Genet,
251(3), 290-297.
Abstract:
Cytochrome P450 gene clusters in Drosophila melanogaster.
Twelve cytochrome P450 cDNA fragments were cloned from Drosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes, Cyp4dl and Cyp4d2, have previously been described. In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of the X chromosome and the other on the left arm of chromosome 2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.
Abstract.
Author URL.
Coustau C, Carkion Y, Nappi A, Shotkoski F, ffrench-Constant R (1996). Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi.
Insect Mol Biol,
5(3), 167-172.
Abstract:
Differential induction of antibacterial transcripts in Drosophila susceptible and resistant to parasitism by Leptopilina boulardi.
Two well-described elements of the immune response of insects include encapsulation of metazoan parasites (blood-cell-mediated) and the production of antibacterial peptides (humoral and/or cellular). However, the possible functional interrelationship between cellular encapsulation and antibacterial responses, and the extent to which the two components may be co-regulated, are poorly understood. We used a novel approach involving strains of Drosophila resistant (R) or susceptible (S) to the wasp parasitoid Leptopilina boulardi to study the expression of three genes involved in the antibacterial response: Dorsal-related immunity factor (Dif), Cecropin (CecA1) and Diptericin (Dip). Both S and R strains produced high levels of all antibacterial transcripts upon bacterial injection. However, when parasitized the R strain showed no induction whilst the S strain did. This lack of antibacterial transcript induction in the parasitized R strain not only clarifies the separation of these two types of immune response but also raises the fascinating possibility of a link in their genetic regulation.
Abstract.
Author URL.
Aronstein K, Auld V, Ffrench-Constant R (1996). Distribution of two GABA receptor-like subunits in the Drosophila CNS.
Invert Neurosci,
2(2), 115-120.
Abstract:
Distribution of two GABA receptor-like subunits in the Drosophila CNS.
Previously we have described the distribution of the Rdl GABA receptor subunit in the Drosophila CNS. Knowing that Rdl can coassemble with LCCH3 (a Drosophila GABA receptor-like subunit showing sequence similarity to vertebrate beta subunit GABAA receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast, Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.
Abstract.
Author URL.
ffrench-Constant R (1996). Ecological and evolutionary aspects of insecticide resistance by John A. McKenzie Academic Press, 1996. £52.00 hbk (185 pages) ISBN 0 12 484825 7. Trends in Ecology & Evolution, 11(9), 391-392.
Shotkoski F, Morris AC, James AA, ffrench-Constant RH (1996). Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter.
Gene,
168(2), 127-133.
Abstract:
Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter.
A single point mutation in the insect gamma-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti. Promoter activity is contained within a 2.53-kb fragment immediately upstream from the rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals anumber of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis-acting control elements underlying ion-channel gene regulation are discussed.
Abstract.
Author URL.
Coustau C, Rocheleau T, Carton Y, Nappi AJ, ffrench-Constant RH (1996). Induction of a putative serine protease transcript in immune challenged Drosophila.
Dev Comp Immunol,
20(4), 265-272.
Abstract:
Induction of a putative serine protease transcript in immune challenged Drosophila.
In an effort to identify serine proteases involved in the insect's immune response, we used a degenerate PCR approach to amplify putative serine protease gene fragments in Drosophila. Sequencing of the cloned PCR products identified one serine protease previously isolated in D. melanogaster (SER1/SER2), as well as two novel putative serine protease gene fragments (SP2, SP3). The involvement of the corresponding genes in the immune response was examined by analyzing their expression in larval mRNA following both parasitic and bacterial exposures. The overexpression of one of the serine proteases-related mRNAs in immune challenged larvae suggests its involvement in the Drosophila immune response.
Abstract.
Author URL.
Hidayat P, Phillips TW, Ffrench-Constant RH (1996). Molecular and Morphological Characters Discriminate Sitophilus oryzae and S. zeamais (Coleoptera: Curculionidae) and Confirm Reproductive Isolation. Annals of the Entomological Society of America, 89(5), 645-652.
Shotkoski F, Zhang HG, Jackson MB, ffrench-Constant RH (1996). Stable expression of insect GABA receptors in insect cell lines. Promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines.
FEBS Lett,
380(3), 257-262.
Abstract:
Stable expression of insect GABA receptors in insect cell lines. Promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines.
We are interested in establishing stably transformed insect cell lines efficiently expressing the insect gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selected marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chlorine ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the generic transformation of non-Drosophilid insects.
Abstract.
Author URL.
Pittendrigh BR, Mocelin G, Andreev O, ffrench-Constant RH (1996). The sequence of a Drosophila Cyp4e2 cytochrome P450-encoding cDNA.
Gene,
179(2), 295-296.
Abstract:
The sequence of a Drosophila Cyp4e2 cytochrome P450-encoding cDNA.
A composite 1458-bp cDNA that encodes cytochrome P450 (P450) Cyp4e2 has been constructed from clones isolated from two Drosophila embryonic cDNA libraries. The Drosophila cDNA open reading frame encodes a protein of 486 amino acids that is 40% identical and 61% similar to Cyp4d1 from Drosophila. The predicted protein is unusual in that it appears to lack the hydrophobic N-terminus typical of microsomal P450s and also contains a small insertion at its C-terminus.
Abstract.
Author URL.
1995
Brun LO, Borsa P, Gaudichon V, Stuart JJ, Aronstein K, Coustau C, ffrench-Constant RH (1995). 'Functional' haplodiploidy. Nature, 374(6522), 506-506.
Brun LO, Stuart J, Gaudichon V, Aronstein K, French-Constant RH (1995). Functional haplodiploidy: a mechanism for the spread of insecticide resistance in an important international insect pest.
Proceedings of the National Academy of Sciences,
92(21), 9861-9865.
Abstract:
Functional haplodiploidy: a mechanism for the spread of insecticide resistance in an important international insect pest.
The coffee berry borer, Hypothenemus hampei, is the most important insect pest of coffee worldwide and has an unusual life history that ensures a high degree of inbreeding. Individual females lay a predominantly female brood within individual coffee berries and because males are flightless there is almost entirely full sib mating. We investigated the genetics associated with this interesting life history after the important discovery of resistance to the cyclodiene type insecticide endosulfan. Both the inheritance of the resistance phenotype and the resistance-associated point mutation in the gamma-aminobutyric acid receptor gene Rdl were examined. Consistent with haplodiploidy, males failed to express and transmit paternally derived resistance alleles. Furthermore, while cytological examination revealed that males are diploid, one set of chromosomes was condensed, and probably nonfunctional, in the somatic cells of all males examined. Moreover, although two sets of chromosomes were present in primary spermatocytes, the chromosomes failed to pair before the single meiotic division, and only one set was packaged in sperm. Thus, the coffee berry borer is "functionally" haplodiploid. Its genetics and life history may therefore represent an interesting intermediate step in the evolution of true haplodiploidy. The influence of this breeding system on the spread of insecticide resistance is discussed.
Abstract.
Stilwell GE, Rocheleau T, ffrench-Constant RH (1995). GABA receptor minigene rescues insecticide resistance phenotypes in Drosophila.
J Mol Biol,
253(2), 223-227.
Abstract:
GABA receptor minigene rescues insecticide resistance phenotypes in Drosophila.
A single point mutation within the GABA receptor gene Resistance to dieldrin (Rdl) confers a high level of resistance to cyclodiene insecticides in a wide range of insects. Previous studies have shown partial rescue of the susceptible phenotype via germline transformation of a 36 kb cosmid coding (or all four alternative Rdl splice forms. Here, we describe the construction of two Rdl promoter/cDNA minigenes, each coding for one of the splice forms alone. Single splice forms rescued both the insecticide susceptible and resistant phenotypes associated with the locus as effectively as the complete cosmid. The minigenes also rescue the lethality associated with homozygous re-arrangements disrupting the Rdl gene, and the level of rescue observed is not increased by the addition of more than one splice form. This demonstrates that only a single Rdl splice form is necessary both to confer insecticide sensitivity and also to rescue lethality. Methods by which phenotype rescue could be enhanced and the potential advantages of using Rdl as a selectable marker are discussed.
Abstract.
Author URL.
Aronstein K, Ffrench-Constant R (1995). Immunocytochemistry of a novel GABA receptor subunit Rdl in Drosophila melanogaster.
Invert Neurosci,
1(1), 25-31.
Abstract:
Immunocytochemistry of a novel GABA receptor subunit Rdl in Drosophila melanogaster.
Following our recent cloning of a novel gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl form cyclodiene resistance locus in Drosophila melanogaster, we were interested in defining its pattern of expression during development. Here we report the raising of an anti-Rdl polyclonal antibody that recognizes a single protein of the expected 65 kDa size in immunoblots of Drosophila head homogenates. In situ hybridization using Rdl cDNA probes and the anti-Rdl antibody shows that Rdl message and protein are highly expressed in the developing central nervous system (CNS) of 15-17 h embryos. Interestingly, despite the use of GABA in both the peripheral and CNS of insects, Rdl GABA receptor subunits appear to be confined to the CNS. Detailed immunocytochemistry of Drosophila brain sections showed particularly strong anti-Rdl antibody staining in the optic lobes, ellipsoid body, fan shaped body, ventrolateral protocerebrum and the glomeruli of the antennal lobes. Results are compared with the distribution of staining observed in the insect CNS with antibodies against GABA itself and synaptotagmin, a synaptic vesicle protein.
Abstract.
Author URL.
Al-Aboodi A, Ffrench-Constant RH (1995). RAPD PCR confirms absence of genetic variation between insecticide resistant variants of the green peach aphid, Myzus persicae (Homoptera: Aphididae). Great Lakes Entomologist., 28(2), 127-133.
Zhang HG, Lee HJ, Rocheleau T, ffrench-Constant RH, Jackson MB (1995). Subunit composition determines picrotoxin and bicuculline sensitivity of Drosophila gamma-aminobutyric acid receptors.
Mol Pharmacol,
48(5), 835-840.
Abstract:
Subunit composition determines picrotoxin and bicuculline sensitivity of Drosophila gamma-aminobutyric acid receptors.
Few gamma-aminobutyric acid (GABA) receptor subunits have been cloned from insects. These include Resistance to dieldrin, or Rdl, and a homologue of the vertebrate GABAA receptor beta subunit. Unlike most vertebrate GABAA receptor subunits, Rdl forms a highly functional homomultimeric receptor. This receptor is picrotoxin (PTX) sensitive but bicuculline (BIC) insensitive and cannot be readily classified within the known GABAA receptor subtypes. In contrast, functional expression of the beta subunit homologue has not been reported. We report that coinfection of cells with recombinant baculoviruses containing Rdl plus beta subunits induces GABA receptors with distinct pharmacological and kinetic properties. Coinfection produces two separate receptor populations: one highly sensitive to PTX but BIC insensitive (Rdl homomultimers) and the other PTX insensitive and BIC sensitive (Rdl plus beta heteromultimers). Putative Rdl plus beta channels also show reduced GABA sensitivity, slow desensitization, rapid bursting, and shorter mean open time. These studies not only localize PTX and BIC sensitivity to two distinct GABA receptor subunits but also demonstrate assembly of two highly divergent GABA receptor subunits. Furthermore, the difference in channel conductance and gating between in vivo and recombinant channels implies the existence of uncharacterized GABA receptor subunits in Drosophila.
Abstract.
Author URL.
1994
Zhang HG, ffrench-Constant RH, Jackson MB (1994). A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.
J Physiol,
479 ( Pt 1)(Pt 1), 65-75.
Abstract:
A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.
1. The Drosophila gene Rdl (resistance to dieldrin) encodes a GABA receptor. An alanine-to-serine mutation in this gene at residue 302 confers resistance to cyclodiene insecticides and picrotoxin. Patch clamp analysis of GABA receptors in cultured neurons from wild type and mutant Drosophila was undertaken to investigate the biophysical basis of resistance. 2. In cultured neurons from both wild type and mutant strains, GABA activated a channel that reversed near 0 mV in symmetrical chloride. GABA dose-response characteristics of wild type and mutant receptors were very similar. 3. GABA responses in neurons from the mutant strains showed reduced sensitivity to the GABA antagonists picrotoxin, lindane and t-butyl-bicyclophosphorothionate. Resistance ratios were 116, 970 and 9 for the three blockers, respectively. Inhibition increased with blocker concentration in a manner consistent with saturation of a single binding site. 4. The mutation reduced the single channel conductance by 5% for inward current and 17% for outward current. The single channel current was approximately 60% lower for outward current than for inward current in both wild type and mutant. 5. Open and closed times were both well fitted by the sum of two exponentials. Resistance was associated with longer open times and shorter closed times, reflecting a net stabilization of the channel open state by a factor of approximately five. 6. The mutation was associated with a marked reduction in the rate of GABA-induced desensitization, and a net destabilization of the desensitized conformation by a factor of 29. 7. The Rdl mutation manifests resistance through two different mechanisms. (a) the mutation weakens drug binding to the antagonist-favoured (desensitized) conformation by a structural change at the drug binding site. (b) the mutation destabilizes the antagonist-favoured conformation in an allosteric sense. The global association of a single amino acid replacement with cyclodiene resistance suggests that the resistance phenotype depends on changes in both of these properties, and that insecticides have selected residue 302 of Rdl for replacement because of its unique ability to influence both of these functions. 8. The location of alanine 302 in the sequence of the Rdl gene product supports a mechanism of action in which convulsants such as picrotoxin bind within the channel lumen, where they induce a rapid conformational change to the desensitized state.
Abstract.
Author URL.
Shotkoski F, Lee HJ, Zhang HG, Jackson MB, ffrench-Constant RH (1994). Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti.
Insect Mol Biol,
3(4), 283-287.
Abstract:
Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti.
We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homomultimeric expression of a gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus-infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100-fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.
Abstract.
Author URL.
Ffrench-Constant RH, Steichen JC, Shotkoski F (1994). Polymerase chain reaction diagnostic for cyclodiene insecticide resistance in the mosquito Aedes aegypti.
Med Vet Entomol,
8(1), 99-100.
Author URL.
Ffrench-Constant RH (1994). The molecular and population genetics of cyclodiene insecticide resistance.
Insect Biochem Mol Biol,
24(4), 335-345.
Abstract:
The molecular and population genetics of cyclodiene insecticide resistance.
Cyclodiene resistance has accounted for over 60% of reported cases of insecticide resistance. Understanding of this resistance can therefore help us answer questions relating to the mechanism and origin of representative resistance-associated mutations, questions fundamental to the molecular and populations genetics of pesticide resistance. The cyclodiene resistance gene Rdl (resistance to dieldrin) was cloned from a mutant of the model insect Drosophila resistant to cyclodienes and picrotoxinin. Rdl codes for a subunit of a novel class of GABA gated chloride ion channels and resistance is correlated with replacement of the same amino acid residue in a wide range of species from different insect orders. This single amino acid replacement Ala302 > Ser, within the proposed lining of the chloride ion channel, also confers insensitivity to the blocking action of cyclodienes and picrotoxinin on GABA gated chloride ion channels expressed in Xenopus oocytes. The resistance mechanism involves both changes in cyclodiene binding site affinity and also a change in the rate of receptor desensitization which destabilizes the cyclodiene-favored conformation. Documentation of the resistance associated mutation has allowed for the design of a PCR based molecular monitoring technique. This technique gives more accurate estimates of resistance gene frequency from smaller sample sizes and has shown the frequency of resistance in apparently unselected populations of Drosophila to be as high as 1%. We are still uncertain as to why resistance persists in the apparent absence of selection pressure and any severe reduction in the fitness of resistant strains, besides a paralytic phenotype at high temperature, remains undocumented.
Abstract.
Author URL.
1993
Ffrench-Constant RH, Rocheleau TA, Steichen JC, Chalmers AE (1993). A point mutation in a Drosophila GABA receptor confers insecticide resistance.
Nature,
363(6428), 449-451.
Abstract:
A point mutation in a Drosophila GABA receptor confers insecticide resistance.
Vertebrates and invertebrates both have GABA (gamma-aminobutyric acid) as a major inhibitory neurotransmitter. GABAA receptors in vertebrates assemble as heteromultimers to form an integral chloride ion channel. These receptors are targets for drugs and pesticides and are also implicated in seizure-related diseases. Picrotoxinin (PTX) and cyclodiene insecticides are GABAA receptor antagonists which competitively displace each other from the same binding site. Insects and vertebrates showing resistance to cyclodienes also show cross-resistance to PTX. Previously, we used a field-isolated Drosophila mutant Rdl (Resistant to dieldrin) insensitive to PTX and cyclodienes to clone a putative GABA receptor. Here we report the functional expression and novel pharmacology of this GABA receptor and examine the functionality of a resistance-associated point mutation (alanine to serine) within the second membrane-spanning domain, the region thought to line the chloride ion channel pore. This substitution is found globally in Drosophila populations. This mutation not only identifies a single amino acid conferring high levels of resistance to the important GABA receptor antagonist PTX but also, by conferring resistance to cyclodienes, may account for over 60% of reported cases of insecticide resistance.
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Author URL.
ffrench-Constant RH, Steichen JC, Rocheleau TA, Aronstein K, Roush RT (1993). A single-amino acid substitution in a gamma-aminobutyric acid subtype a receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations.
Proc Natl Acad Sci U S A,
90(5), 1957-1961.
Abstract:
A single-amino acid substitution in a gamma-aminobutyric acid subtype a receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations.
Resistance to cyclodiene insecticides, documented in at least 277 species, is perhaps the most common kind of resistance to any pesticide. By using cyclodiene resistance to localize the responsible gene, a gamma-aminobutyric acid type a receptor/chloride ion-channel gene was previously cloned and sequenced from an insecticide-susceptible Drosophila melanogaster strain. We now describe the molecular genetics of the resistance allele. A single-base-pair mutation, causing a single-amino acid substitution (Ala-->Ser) within the second membrane-spanning region of the channel, was found to be the only consistent difference between resistant and susceptible strains of D. melanogaster. Some resistant strains of Drosophila simulans show the same mutation, whereas others show an alternative single-base-pair mutation in the same codon, resulting in the substitution of a different amino acid (glycine). These constitute single-box-pair mutations in insects that confer high levels of resistance to insecticides. The presence of the resistance mutations was then tested in a much larger set of strains by the PCR and subsequent digestion with a diagnostic restriction endonuclease. Both resistance-associated mutations cause the loss of a Hae II site. This site was invariably present in 122 susceptible strains but absent in 58 resistant lines of the two species sampled from five continents. PCR/restriction endonuclease treatment was also used to examine linkage of an EcoRI polymorphism in a neighboring intron in D. melanogaster, which was found associated with resistance in all but 3 of 48 strains examined. These PCR-based techniques are widely applicable to examination of the uniqueness of different resistance alleles in widespread populations, the identification of resistance mechanisms in different species, and the determination of resistance frequencies in monitoring.
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Thompson M, Shotkoski F, ffrench-Constant R (1993). Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti. Conservation of the gene and resistance associated mutation with Drosophila.
FEBS Lett,
325(3), 187-190.
Abstract:
Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti. Conservation of the gene and resistance associated mutation with Drosophila.
In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila. In D. melanogaster, resistance to cyclodienes and picrotoxinin is caused by a single amino acid substitution (alanine to serine) in the putative channel lining of a gamma-aminobutyic acid gated chloride ion channel. We report that the mosquito gene not only shows high homology to that of Drosophila but also that resistant strains display substitution of the same amino acid. The significance of this result in relation to the evolution of pesticide resistance, the use of Drosophila as a model insect for resistance studies and the potential use of this gene as a selectable marker in the genetic transformation of non-Drosophilids is discussed.
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Author URL.
ffrench-Constant RH (1993). Cloning of a putative GABAA receptor from cyclodiene-resistant Drosophila: a case study in the use of insecticide-resistant mutants to isolate neuroreceptors. In (Ed) Comparative Molecular Neurobiology, Springer Nature, 210-223.
ffrench-Constant RH (1993). Cloning of a putative GABAA receptor from cyclodiene-resistant Drosophila: a case study in the use of insecticide-resistant mutants to isolate neuroreceptors.
EXS,
63, 210-223.
Abstract:
Cloning of a putative GABAA receptor from cyclodiene-resistant Drosophila: a case study in the use of insecticide-resistant mutants to isolate neuroreceptors.
This chapter uses the isolation and cloning of cyclodiene resistance from Drosophila melanogaster to illustrate how mutants resistant to a toxicant can be used to study neuroreceptors. Isolation of mutants from the field, mapping of the single gene responsible and its subsequent cloning are described. As confirmation of gene cloning a susceptible allele of the gene has been used to genetically transform resistant individuals to susceptibility. The gene product appears to code for a subunit of a receptor highly similar to vertebrate GABAA receptor/chloride ion channels, and functional expression studies are described which will elucidate its pharmacology. Cyclodiene resistance is extremely widespread, occurring in both invertebrates and vertebrates. Thus examination of resistance-associated mutations in this receptor in a range of species will enhance our understanding of both the binding sites of toxic ligands and the genetic basis of pesticide resistance.
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Ffrench-Constant RH (1993). Cloning of the Drosophila cyclodiene insecticide resistance gene: a novel GABAA receptor subtype?.
Comp Biochem Physiol C Comp Pharmacol Toxicol,
104(1), 9-12.
Abstract:
Cloning of the Drosophila cyclodiene insecticide resistance gene: a novel GABAA receptor subtype?
1. gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in both vertebrates and invertebrates. GABAA receptors are composed of a number of different subunits that assemble to form a chloride ionophore. 2. Several subunit types, alpha, beta, gamma, delta and rho have been cloned from vertebrates, but until recently these receptors have remained uncloned from invertebrates. 3. GABAA receptors form the proposed site of action of cyclodiene insecticides. Therefore a Drosophila mutant (Rdl), resistant to cyclodienes and the GABAA receptor ligand picrotoxin (PTX), was used to clone the gene responsible for resistance as a putative invertebrate GABAA receptor. 4. Analysis of the predicted amino acid sequence and gene structure shows that Rdl codes for a receptor subunit similar to vertebrate GABAA receptors, but sufficiently different that it may represent a novel class of GABAA receptor subtype. 5. Cyclodiene insecticide resistance accounts for over 60% of reported cases of insecticide resistance and is also found in vertebrates. 6. Therefore elucidating the molecular basis of cyclodiene resistance is not only important to our understanding of pesticide action on the GABAA receptor but also in examining the conservation of the resistance mechanism between vertebrates and invertebrates.
Abstract.
Author URL.
Thompson M, Steichen JC, ffrench-Constant RH (1993). Conservation of cyclodiene insecticide resistance-associated mutations in insects.
Insect Mol Biol,
2(3), 149-154.
Abstract:
Conservation of cyclodiene insecticide resistance-associated mutations in insects.
Cyclodiene insecticide resistance has accounted for over 60% of reported cases of insecticide resistance. In Drosophila melanogaster resistance is associated with a single base pair substitution in the GABA receptor/chloride ion channel gene Rdl. This substitution predicts the replacement of an alanine with a serine in the second membrane spanning domain, the region thought to line the chloride ion channel pore. Here we report, via the use of degenerate primers in the polymerase chain reaction, that precisely the same substitution is present in three pests from three different insect orders: the house fly (Diptera), red flour beetle (Coleoptera) and American cockroach (Dictyoptera). This finding suggests that there are a limited number of mutations that can confer resistance to cyclodienes, putative channel blockers, while still maintaining adequate chloride ion channel function. The conservation of the resistance-associated mutation between Drosophila and pest insects directly validates the approach of using this insect as a model system for isolating and studying resistance genes. The importance of single base pair substitutions in the evolution of pesticide resistance and in the design of molecular monitoring techniques is discussed.
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ffrench-Constant RH, Roush RT, Cariño FA (1993). Drosophila as a Tool for Investigating the Molecular Genetics of Insecticide Resistance. In (Ed) Molecular Approaches to Fundamental and Applied Entomology, Springer Nature, 1-37.
ffrench-Constant RH, Rocheleau TA (1993). Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing.
J Neurochem,
60(6), 2323-2326.
Abstract:
Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing.
The Drosophila gamma-aminobutyric acid (GABA) receptor subunit gene Rdl was isolated on the basis of a mutant phenotype showing high levels of insensitivity to picrotoxinin and cyclodiene insecticides. Following analysis of two dissimilar cDNAs isolated from the locus, we report that Rdl undergoes extensive alternative splicing at two locations in the putative extracellular domain. At each location a choice is made between exons of the same size: "a" or "b" (23 amino acids long with two substitutions) and "c" or "d" (46 residues long with 10 substitutions). The function of these alternative exons remains unclear; however, exon d contains a putative site for casein kinase II phosphorylation. All possible combinations of exons (a with c or d and b with c or d) were found in RNA isolated from early embryos. This is the first demonstration of alternative splicing in a GABA receptor gene from invertebrates.
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Lee HJ, Rocheleau T, Zhang HG, Jackson MB, ffrench-Constant RH (1993). Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl.
FEBS Lett,
335(3), 315-318.
Abstract:
Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl.
Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-Rdl antiserum recognized a protein of the expected size of approximately 65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 microM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (approximately 100-fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.
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1992
Ffrench-Constant RH, Roush RT (1992). Cloning of a locus associated with cyclodiene resistance in Drosophila: a model system in a model insect.
ACS symposium series.,
505, 90-98.
Abstract:
Cloning of a locus associated with cyclodiene resistance in Drosophila: a model system in a model insect
A locus associated with cyclodiene resistance has been cloned from Drosophila. A strain showing high levels of resistance to cyclodienes was isolated from the field and the gene mapped to the polytene subregion 66F on the left arm of chromosome three. The gene was cloned following a cosmid walk across the region and identification of several inversion breakpoints uncovering resistance. A number of cDNAs have been isolated from the locus. Sequencing of one of these showed high homology to vertebrate GABA(A) subunits. The susceptible phenotype has been rescued following P-element mediated germline transformation of a cosmid containing the cloned susceptible gene. The use of the cloned gene to study gene dosage, protein expression, and identification of the resistance associated mutation is discussed. Functional expression studies are described to determine the precise nature of the receptor.
Abstract.
Ffrench-Constant RH, Roush RT (1992). Cloning of an Invertebrate GABA Receptor from the Cyclodiene Resistance Locus in Drosophila. In (Ed) Neurotox ’91, Springer Nature, 285-292.
ffrench-Constant RH, Rocheleau T (1992). Drosophila cyclodiene resistance gene shows conserved genomic organization with vertebrate gamma-aminobutyric acidA receptors.
J Neurochem,
59(4), 1562-1565.
Abstract:
Drosophila cyclodiene resistance gene shows conserved genomic organization with vertebrate gamma-aminobutyric acidA receptors.
Genomic clones from the Rdl locus of Drosophila, whose mutant phenotype is resistant to cyclodiene insecticides and picrotoxin, were characterized by restriction mapping and partial sequencing to determine intron/exon structure. The coding region of the gene comprises nine identified exons and spans greater than 25 kb of genomic DNA. The structure of the Drosophila Rdl receptor subunit was compared with those of vertebrate gamma-aminobutyric acid subtype a (GABAA) receptors and nicotinic acetylcholine receptors (nAChRs). The first six introns in Rdl show positions similar to those in vertebrate GABAA receptors, whereas the last two differ. It is interesting that the last intron appears to be in a position similar to that in nAChRs. These results are examined in relation to the proposal, based on amino acid identities, that Rdl codes for a novel class of GABAA receptor subunit more closely related to glycine receptors, and the possible place of Rdl in the lineage of the receptor superfamily is discussed.
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Bloomquist JR, Roush RT, ffrench-Constant RH (1992). Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).
Arch Insect Biochem Physiol,
19(1), 17-25.
Abstract:
Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).
Toxicological and neurophysiological studies were performed to characterize the resistance mechanism in a cyclodiene-resistant strain of Drosophila melanogaster (Maryland strain). Dieldrin had an LC50 of 0.058 ppm against the larvae of susceptible D. melanogaster (Oregon-R wild type) when formulated in the rearing media. The LC50 of the resistant Maryland strain was 10.8 ppm, giving a resistance ratio (LC50-Maryland/LC50-susceptible) of 186-fold. Suction electrode recordings were made from peripheral nerves of the larval central nervous system to test whether reduced nerve sensitivity played any role in the observed resistance. In susceptible preparations (n = 5), inhibition of nerve firing by 1 mM gamma-aminobutyric acid (GABA) was effectively antagonized within 3-10 min by 10 microM dieldrin. In contrast, 30 min incubations with 10 microM dieldrin had no effect on preparations from cyclodiene-resistant individuals (n = 5). Similarly, 10 microM picrotoxinin blocked GABA-dependent inhibition in susceptible nerve preparations (n = 3). In recordings from resistant insects (n = 4), picrotoxinin displayed either weak antagonism of GABA or hyperexcitation indistinguishable from susceptible preparations. These results demonstrate that cyclodiene resistance in the Maryland strain of D. melanogaster 1) is expressed in immature stages, 2) is present at the level of the nerve, and 3) extends to picrotoxinin, albeit at a reduced level compared with dieldrin. The possible role of an altered GABA receptor in this resistance is discussed.
Abstract.
Author URL.
ffrench-Constant RH, Aronstein K, Roush RT (1992). Use of a P-element mediated germline transformant to study the effect of gene dosage in cyclodiene insecticide-resistant Drosophila melanogaster (Meigen). Pesticide Biochemistry and Physiology, 43(1), 78-84.
1991
Ffrench-Constant RH, Roush RT (1991). Gene mapping and cross-resistance in cyclodiene insecticide-resistant Drosophila melanogaster (Mg.).
Genet Res,
57(1), 17-21.
Abstract:
Gene mapping and cross-resistance in cyclodiene insecticide-resistant Drosophila melanogaster (Mg.).
Resistance to the cyclodiene insecticide dieldrin maps to a single gene (Rdl) on the left arm of chromosome III in Drosophila melanogaster (Meigen). The gene was further mapped by the use of chromosomal deficiencies to a single letter sub-region, 66F, on the polytene chromosome. The cross-resistance spectrum of a backcrossed strain lacking elevated mixed function oxidase activity, a common resistance mechanism, was examined. Levels of resistance similar to those found in other insects were found to dieldrin, aldrin, endrin, lindane, and picrotoxinin. Strong similarity of this single major gene with that found in other cyclodiene resistant insects is suggested by its cross-resistance spectrum and chromosomal location, via homology with other Diptera. The significance of major genes in insecticide resistance is discussed.
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Ffrench-Constant RH, Mortlock DP, Shaffer CD, MacIntyre RJ, Roush RT (1991). Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype a receptor locus.
Proc Natl Acad Sci U S A,
88(16), 7209-7213.
Abstract:
Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype a receptor locus.
Cyclodiene resistance represents 60% of the reported cases of insecticide resistance and is also present in vertebrates. Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype a (GABAA) receptor-chloride ionophore complex. Following isolation of cyclodiene-resistant Drosophila mutants, we report the cloning of the locus conferring resistance via a "chromosomal walk" and rescue of the susceptible phenotype by P-element-mediated germ-line transformation. Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits. To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation. This gene may be useful as a selectable marker in other insect systems.
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1990
Ffrench-Constant RH, Roush RT, Mortlock D, Dively GP (1990). Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae).
J Econ Entomol,
83(5), 1733-1737.
Abstract:
Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae).
High levels (about 4,000-fold) of resistance to dieldrin were isolated by screening field-collected populations of Drosophila melanogaster (Meigen). The resistance was made homozygous following 2-4 generations of selection. A single, major gene mapping to the left arm of chromosome III was solely responsible for resistance. The implications of the recovery of resistant mutants from field populations of D. melanogaster are discussed.
Abstract.
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ffrench-Constant RH, Roush RT, MacIntyre RJ (1990). Isolation, Characterization and Progress in Cloning of Cyclodiene Insecticide Resistance in Drosophila melanogaster. In (Ed) Molecular Insect Science, Springer Nature, 41-48.
ffrench-Constant RH, Roush RT (1990). Resistance Detection and Documentation: the Relative Roles of Pesticidal and Biochemical Assays. In (Ed) Pesticide Resistance in Arthropods, Springer Nature, 4-38.
Lines JD, ffRench-Constant RH, Kasim SH (1990). Testing Anopheles albimanus for genetic linkage of insecticide resistance genes by combining insecticide bioassay and biochemical methods.
Med Vet Entomol,
4(4), 445-450.
Abstract:
Testing Anopheles albimanus for genetic linkage of insecticide resistance genes by combining insecticide bioassay and biochemical methods.
A microtitre-plate assay which distinguishes propoxur-resistant from susceptibles Anopheles albimanus Weidemann was used to test for linkage between the genes for propoxur- and dieldrin-resistance. The adult progeny of a backcross between a doubly-resistant colony and a fully susceptible colony were exposed in conventional test kits to the standard discriminating dose of dieldrin, and kept in the insectary overnight. Both live and dead insects were then assayed individually for propoxur-resistance. The results showed that heterozygotes for propoxur-resistance could be reliably distinguished from susceptibles whether or not they had been killed up to 24 h previously by dieldrin treatment. In this way all the backcross progeny could be scored at both resistance loci, and all four genotypic classes identified. Resistant and susceptible alleles at the two loci were inherited independently, demonstrating the absence of linkage. The usual method of testing for linkage between resistance genes is inefficient and open to bias, because insects have to be exposed to each insecticide in turn, and only half of them can be scored at both loci. The method shown here avoids these drawbacks.
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1989
Ffrench-Constant RH, Bonning BC (1989). Rapid microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotypes in the mosquitoes Anopheles albimanus, An. nigerrimus and Culex pipiens.
Med Vet Entomol,
3(1), 9-16.
Abstract:
Rapid microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotypes in the mosquitoes Anopheles albimanus, An. nigerrimus and Culex pipiens.
A rapid method of distinguishing insecticide insensitive acetylcholinesterase (AChE) genotypes was applied to three species of mosquitoes. This relies on comparing rates of an AChE mediated reaction in the presence and absence of insecticides which are inhibitors, using a kinetic microtitre plate reader. Clearer and more rapid resolution between genotypes was achieved than with previous assays which measure the amount of product formed at a fixed end-point. Results are presented for the F1s from crossing resistant and susceptible Anopheles albimanus Wiedemann and Culex pipiens L. for a strain of An. albimanus with a translocation linking the AChE gene to the Y chromosome and for field collected An. nigerrimus Giles. Propoxur and malaoxon were used as inhibitors. In all three species the enzyme was more insensitive to propoxur than malaoxon. Susceptible enzymes in all species also showed higher uninhibited AChE activity than their resistant counterparts. Presentation of both inhibited and uninhibited activities side by side may be useful to identify insects likely to be misclassified due to abnormally low AChE activities. Estimated frequencies of the three resistance genotypes in field populations of An. nigerrimus conformed to Hardy-Weinberg ratios. The implications of this technique for laboratory and field studies on insects are discussed.
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Harrington R, Bartlet E, Riley DK, ffrench-Constant RH, Clark SJ (1989). Resurgence of insecticide-resistant Myzus persicae on potatoes treated repeatedly with cypermethrin and mineral oil. Crop Protection, 8(5), 340-348.
Field LM, Devonshire AL, ffrench-Constant RH, Forde BG (1989). The combined use of immunoassay and a DNA diagnostic technique to identify insecticide-resistant genotypes in the peach-potato aphid, Myzus persicae (Sulz.). Pesticide Biochemistry and Physiology, 34(2), 174-178.
1988
ffrench-Constant RH, Harrington R, Devonshire AL (1988). Effect of repeated applications of insecticides to potatoes on numbers of Myzus persicae (Sulzer) (Hemiptera: Aphididae) and on the frequencies of insecticide-resistant variants. Crop Protection, 7(1), 55-61.
French-Constant RH, Byrne FJ, Stribley MF, Devonshire AL (1988). Rapid identification of the recently recognised Myzus antirrhinii (Macchiati) (Hemiptera: Aphididae) by polyacrylamide gel electrophoresis. Entomologist., 107(1), 20-23.
Ffrench-Constant RH, Devonshire AL, White RP (1988). Spontaneous loss and reselection of resistance in extremely resistant Myzus persicae (Sulzer). Pesticide Biochemistry and Physiology, 30(1), 1-10.
1987
ffrench-Constant RH, Devonshire AL (1987). A multiple homogenizer for rapid sample preparation in immunoassays and electrophoresis.
Biochem Genet,
25(7-8), 493-499.
Abstract:
A multiple homogenizer for rapid sample preparation in immunoassays and electrophoresis.
A multiple homogenizer is described for preparing samples of small invertebrates or tissue in a flat-bottom immunoplate. Its efficiency was evaluated by immunoassay of carboxylesterase (E4), the enzyme conferring insecticide resistance in the peach potato aphid (Myzus persicae). This equipment was shown to release more enzyme, with less variability, than homogenizing individual aphids and its efficiency allows one person to analyze up to 3000 individual insects per day. It is also suitable for preparing samples for electrophoretic analysis. In the present study samples were loaded onto electrophoresis gels rapidly and accurately by using an eight-channel multipipette.
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1986
Ffrench-Constant RH, Devonshire AL (1986). Effect of different insecticides on the selection and control of highly resistant Myzus persicae.
Ffrench-Constant RH, Devonshire AL (1986). The effect of aphid immigration on the rate of selection of insecticide resistance in Myzus persicae by different classes of insecticides. Aspects of applied biology(13), 115-125.
